Artificial beta cells and methods of use thereof

ABSTRACT

Disclosed herein is a particle containing an inner liposomal vesicle (ILV) encapsulating a therapeutic agent; an outer liposomal vesicle (OLV) encapsulating the ILV; a membrane fusion-promoting agent; and a pH-altering agent. Also disclosed are methods of delivering a therapeutic agent to a subject comprising: a) providing a herein disclosed particle b) triggering ILV and OLV fusion; and c) releasing the therapeutic agent outside of the OLV. Also disclosed are methods for treating a disease in a subject in need thereof comprising: administering to a subject a herein disclosed particle. Also disclosed are methods to release insulin to an environment comprising increased glucose levels, the method comprising exposing to the environment a herein disclosed particle.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application Serial No.16/648,026, filed on Mar. 17, 2020, which is a national stageapplication of PCT International Application No. PCT/US2018/051310,filed on Sep. 17, 2018, entitled “ARTIFICIAL β-CELLS AND METHODS OF USETHEREOF,” which claims the benefit of U.S. Provisional Pat. ApplicationSerial No. 62/559,909, filed Sep. 18, 2017, all of which areincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing encoded as ASCIItext which was filed electronically by EFS-web and is herebyincorporated by reference in its entirety. Said ASCII text copy of theSequence Listing, created on Jul. 13, 2022, is named “10620-050WO12022_07_13 ST26.XML” and is 13,400 bytes in size.

FIELD

The disclosure herein relates to particles, methods for delivering atherapeutic agent, and methods for treating a disease (e.g., diabetes).

BACKGROUND

Pancreatic β-cells dynamically regulate insulin secretion to maintainblood glucose homeostasis. Destruction or dysfunction of these cellsleads to type 1 and type 2 diabetes mellitus, a family of chronicdiseases that currently affect over 415 million people in the world(Rorsman, P., Annu. Rev. Physiol. 75, 155-179 (2013); Yu, J. et al.,Proc. Natl Acad. Sci. USA 112, 8260-8265 (2015); Ohkubo, Y. et al.,Diabetes. Res. Clin. Pract. 28, 103-117 (1995)). Inadequate glucosecontrol caused by loss of β-cell function can lead to hyperglycemia,which is directly implicated in the development of severe complications,including blindness, renal failure and cardiovascular disease (Nathan,D., New Engl. J. Med. 328, 1676-1685 (1993)). On the other hand,traditional intensive insulin therapy by periodic injections imperfectlysimulates the dynamics of β-cells and can cause hypoglycemia, which isassociated with risks of behavioral and cognitive disturbance, braindamage, or death (Nathan, D., Diabetes Care 37, 9-16 (2014); Control, T.et al., New Engl. J. Med. 329, 977-986 (1993); Orchard, T. et al., JAMA313, 45-53 (2015)). Cell therapy is gathering momentum as a promisingstrategy for restoring tight glycemic control while mitigating episodesof both hyper- and hypoglycemia in patients with diabetes (Xie, M. etal., Science 354, 1296-1301 (2016); Pepper, A. et al., Nat. Biotech. 33,518-523 (2015); Vegas, A.J. et al., Nat. Med. 22, 306-311 (2016)). Yetthis approach is limited by the shortage of donor islets and therequirement for immunosuppression after transplantation (Veiseh, O. etal., Nat. Rev. Drug. Discov. 14, 45-57 (2015)).

As an alternative to the use of living cells for therapeutic purposes, avariety of biomimetic assemblies have been proposed to recreate the keyfunctions of cells (Zhang, Y. et al., Trends Biotechnol. 26, 14-20(2008); Szostak, J. et al., Nature 409, 387-390 (2001)). Prominentexamples of complex assemblies include cell membrane-cloakednanoparticles, deformable microgels and vesicles with integratedproteins for detoxification, vaccines, haemostasis and drug release (Hu,C. et al., Nat. Nano. 8, 933-938 (2013); Hu, C. et al., Nature 526,118-121 (2015); Brown, A.C. et al., Nat. Mater. 13, 1108-1114 (2014);Hu, Q. et al., Adv. Mater. 28, 9573-9580 (2016); Molinaro, R. et al.,Nat. Mater. 15, 1037-1046 (2016)). All of these assemblies can be viewedas single-compartment structures, and their interactions with biologicalentities are relatively passive compared to those exhibited by ‘living’cells. Although multi-compartmentalized assemblies have been created toreconstitute the hierarchical architecture of cells for delivering drugcocktails or conducting cascade reactions (Boyer, C. et al., ACS Nano 1,176-182 (2007); Wong, B. et al., Adv. Mater. 23, 2320-2325 (2011);Marguet, M. et al., Angew. Chem. Int. Ed. 51, 1173-1176 (2012); Peters,R. et al., Angew. Chem. Int. Ed. 53, 146-150 (2014); Elani, Y. et al.,Nat. Commun. 5, 5305-5309 (2014); Chiu, H. et al., Angew. Chem. Int. Ed.47, 1875-1878 (2008)), replication of “sense-react” behaviors of naturalcells remains elusive. Indeed, a key challenge in the de novo design ofsynthetic therapeutic cells is to mimic the higher-order functions oftheir natural counterparts that can precisely sense the externalenvironment, make an internal decision, and release feedback (Zhang, Y.et al., Trends Biotechnol. 26, 14-20 (2008); Lu, Y., et al., Nat. Rev.Mater. 1, 16075 (2016)), a process routinely employed by pancreaticβ-cells in response to changes in glycemic levels.

Like other biomolecule-secreting cells, β-cells can export cellularcargos to the outside through a vesicle transport system and a membranefusion process upon external stimulation (Hata, Y., et al., Nature 366,347-351 (1993); Kaiser, C. et al., Cell 61, 723-733 (1990); Sollner, T.et al., Nature 362, 318-324 (1993)).

The compositions and methods disclosed herein address these and otherneeds.

SUMMARY

Disclosed herein is a novel particle having a sense-and-response systemfor the delivery of a therapeutic agent (e.g., insulin).

Disclosed herein is a particle comprising: an inner liposomal vesicle(ILV) encapsulating a therapeutic agent; an outer liposomal vesicle(OLV) encapsulating the ILV; a membrane fusion-promoting agent; and apH-altering agent. In some aspects, the therapeutic agent is insulin. Insome aspects, the membrane fusion-promoting agent promotes fusionbetween the ILV and the OLV. In some aspects, the pH-altering agentcomprises a glucose-responsive enzyme. In some aspects, the ILV furthercomprises a membrane fusion-inhibiting agent which shields access to themembrane fusion-promoting agent.

Also disclosed herein are methods of delivering a therapeutic agent to asubject comprising: a) providing a particle comprising an innerliposomal vesicle (ILV) encapsulating a therapeutic agent, an outerliposomal vesicle (OLV) encapsulating the ILV, a membranefusion-promoting agent, and a pH-altering agent; b) triggering ILV andOLV fusion; and c) releasing the therapeutic agent outside of the OLV.In some aspects, the triggering step b) is facilitated by binding of themembrane fusion-promoting agent. In some aspects, the releasing c) isfacilitated in hyperglycemic conditions. In some aspects, the releasingc) is inhibited in hypoglycemic conditions.

Also disclosed herein are methods of treating a disease in a subject inneed thereof comprising: administering to a subject a particlecomprising an inner liposomal vesicle (ILV) encapsulating a therapeuticagent, an outer liposomal vesicle (OLV) encapsulating the ILV, amembrane fusion-promoting agent, and a pH-altering agent. In someaspects, the disease is diabetes. In some aspects, the particle treatshyperglycemia and avoids inducing hypoglycemia.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

Figure 1 shows the design and synthesis of the artificial β-cell (AβC).GLUT2, GOx and CAT are abbreviations for glucose transporter 2, glucoseoxidase, and catalase, respectively. a, Schematic summation of thebiochemical processes inside the AβCs. b, (Top) The inner smallliposomal vesicles (ISVs), stained with uranyl-acetate and imaged bytransmission electron microscopy, were used to mimic insulin granulesinside natural β cells (scale bar, 100 nm). (Bottom) showed the sizedistribution histogram of the insulin-containing ISVs. c, Cryo-SEM (Top)and size distribution (Bottom) and d, magnified fractured cryo-SEM ofthe vesicles-in-vesicle superstructures (scale bar in c, 5 µm; scale barin d, 200 nm). From the fracture in panel d, small liposomal vesiclescan be clearly seen inside the large liposome. e, Confocal lasermicroscopy image (CLSM) to verify the encapsulation of glucose oxidaselabeled with fluorescein isothiocyanate (GOx-FITC) and catalase labeledwith rhodamine B (CAT-RB) inside the large liposomes (scale bar, 5 µm).f, Western blotting results indicating the retention of immunoreactivityof glucose transporter 2 (GLUT2) in the superstructures. g, CLSM imageshowing the reconstitution of GLUT2 labeled with RB on the membranes ofthe larger liposomes (scale bar, 5 µm). h, CLSM image to demonstrate theinsertion of the proton channel gramicidin A labeled withlysine-5-carboxyfluorescein into the outer membrane (scale bar, 5 µm).

FIGS. 2A-2I is a set of graphs and schematics depicting biochemicalprocesses inside AβCs. FIGS. 2A-C, Glucose sensing ability of AβCs. pHvariation inside AβCs in 400 (FIG. 2A) and 100 (FIG. 2B) mg/dL glucosesolutions. The ratios represent the relative amount of gramicidin A tothat of dipalmitoylphosphatidylcholine lipid. FIG. 2C, Reversible pHvariation induced by alternatively switching environmental glucoseconcentrations. FIGS. 2D-2I, ‘Signal transduction’ inside AβCs tocontrol PEG deshielding and peptide assembly. FIG. 2D, Schematic forreversible PEG association and disassociation tuned by glucosemetabolism. PEG-CDNA was labeled with the tetramethylrhodamine(DNA-donor), while GDNA-CH was labeled with IabRQ (DNA-acceptor). FIG.2E, FRET assay to study the dehybridization of the pH-sensitive DNAduplex which bridges the PEG shield and the ISV surface at differentglucose concentrations. FIG. 2F, Reversible quenching and recovery ofthe fluorescence of DNA-donor to prove the reversible attachment anddetachment of the PEG shield in high and low glucose solutions. FIG. 2G,Schematic illustration of the quenching of nitrobenzofuran (NBD,peptide-donor) on peptide-K by tetramethylrhodamine (TRMRA,peptide-acceptor) on peptide-E induced by peptide assembly after PEGdissociation. FIG. 2H, FRET assay to study the interactions of peptide-Eand peptide-K at different glucose concentrations. In FIG. 2E and FIG.2H, F₀ and F_(t) represent the fluorescence intensity measured beforeand at time t after addition into glucose solutions; inset: (I) and (II)respectively show the fluorescence spectra of the AβCs before and afterincubation in 400 mg/dL glucose solution. FIG. 2I, Step decrease influorescence intensity of peptide-donor by switching the glucoseconcentrations. Data points represent mean ± SD (n = 3).

FIGS. 3A-3E shows membrane fusion of the ISV and OLV following glucosemetabolism. FIG. 3A, Fluorescence spectra of the AβCs after incubatingin 400 mg/dL glucose solution for different time. The ISV weresimultaneously labeled with lipid-donor (NBD, emission λ_(max)=536 nm)and lipid-acceptor (LR, emission λ_(max)=592 nm). Total lipid-donorfluorescence spectrum was obtained by destructing AβCs with 1% TritonX-100. FIG. 3B, Kinetics profiles of lipid mixing betweenlipid-donor/acceptor-labeled ISV and OLV in 400 mg/dL and 100 mg/dlglucose solutions, as indicated by the increase in lipid-donor emission.Each data point represents an average of triplicate measurements withstandard error <10%. CLSM images showed the lipid mixing betweenlipid-donor/acceptor-labeled ISV and OLV in FIG. 3C, 400 mg/dL and FIG.3D, 100 mg/dL glucose solutions after different incubation time (scalebar, 1 µm). The profiles in FIG. 3C and FIG. 3D showed the distributionof the fluorescence intensity of lipid-donor (green lines) andlipid-acceptor (red lines) along the indicated white dash lines. FIG.3E, Step increase in the fluorescence of lipid-donor after alternativelychanging the glucose concentrations between 400 and 100 mg/dL. Datapoints represent mean ± SD (n = 3).

FIGS. 4A-4G shows in vitro insulin ‘secretion’ from AβCs and in vivotype-1 diabetes treatment. FIG. 4A, In vitro accumulated insulin releasefrom AβCs incubated in solutions with different glucose concentrations.Data points represent mean ± SD (n = 3). FIG. 4B, 2.5 D CLSM imagesshowed the fluorescence intensity and distribution of fluoresceinlabeled-insulin from AβCs before and after incubation in solutionscontaining different concentrations of glucose. FIG. 4C, Pulsatilerelease profile by AβCs presents the insulin release rate upon switchingthe glucose concentrations between 400 and 100 mg/dL. Data pointsrepresent mean ± SD (n = 3). FIG. 4D, The blood glucose levels ofdiabetic mice after ‘transplantation’ with AβCs, or control AβCs whichlacked insulins (AβC_((no) _(insulin))), membrane fusionpeptide-E/peptide-K (AβC_((no) _(PE/PK))), or glucose sensing machinery(AβC_((no) _(GSM))). Data points represent mean ± SD (n = 5). *P<0.05for AβCs compared with control AβCs. FIG. 4E, Blood glucose levels werecontinuously monitored in the first 12 h shown in (FIG. 4D). Data pointsrepresent mean ± SD (n = 5). FIG. 4F, Variation of plasma insulinconcentration in diabetic mice over time after transplantation of AβC orcontrol AβCs. Data points represent mean ± SD (n = 5). FIG. 4G, In vivointraperitoneal glucose tolerance test (IPGTT) performed toward diabeticmice at 24, 36, and 48 h following AβC treatment in comparison to thehealthy control mice. Data points represent mean ± SD (n = 5).

FIG. 5 is a schematic depicting the design and synthesis of theartificial β-cell. GOx, CAT, ISV and OLV are abbreviations for glucoseoxidase, catalase, inner small vesicle and outer large vesicle,respectively.

FIG. 6 is a DNA gel electrophoresis photograph. a, Agarose (2%)electrophoresis of DNA ladder (lane 1), cytosine-rich DNA (CDNA, lane 2)and PEG₅₀₀₀ conjugated CDNA (lane 3); b, Agarose (2%) electrophoresis ofDNA-ladder (lane 1), control DNA1 (lane 2) and PEG₅₀₀₀ conjugated DNA1(lane 3).

FIG. 7 is a set of graphs depicting the CD spectra of PEG-CDNA/GDNA-CH(a) and PEG-DNA1/DNA2-CH (c) that illustrate DNA conformational changesat different pH’s. b, the CD intensity of the characteristic band oftetraplex DNA (summation of i-motif and G-quadruplex) around 287 nmversus different pH values while (d) shows the CD intensity of thecharacteristic band of duplex DNA at 278 nm versus pH values. From theresults, it can be concluded that PEG-CDNA/GDNA-CH dehybridized andformed tetraplexes below pH 5.5 while the control PEG-DNA1/DNA2-CHshowed no dissociation during the studied pH range. Data pointsrepresent mean ± SD (n = 3).

FIG. 8 is a set of graphs depicting reversible, pH-controlledattachment/detachment of PEG shield on the ISV surface studied using afluorescent resonance energy transfer (FRET) assay. Fluorescent spectraof (a) PEG₅₀₀₀-conjuagted C-rich DNA (labeled with tetramethylrhodamine,DNA-donor)/cholesterol-ended G-rich DNA (labeled with IAbRQ,DNA-acceptor)-inserted ISVs and (b) PEG₅₀₀₀-DNA1 (labeled withDNA-donor)/DNA2 (labeled with DNA acceptor)-cholesterol-inserted ISVs atpH 7.4 and pH 5.5 with excitation at 555 nm. See Table 1 for DNAsequence details. (c) and (d), respectively showed the fluorescenceintensity of DNA-donor in (a) and (b) at different cycles of switchingpH at 7.4 and 5.5. The fluorescence intensity at each cycle was measured20 min after switching the pH. Data points represent mean ± SD (n = 3).

FIG. 9 shows a, sequences of peptide-K and peptide-E with threecomponents-a transmembrane domain, a spacer and a recognition motif,mimicking the membrane fusion SNARE polypeptides (SNARE = soluble NSFattachment protein receptor). The sequences were designed by modifyingpreviously reported peptides. b, CD spectra of peptide-E, peptide-K, andpeptide-E/peptide-K. Peptide-K shows substantial α-helical content in CDspectra ([θ]_(220 nm)/[θ]_(208 nm) is ~0.71), whereas peptide-E adopts apredominantly random coil structure. After mixing, the α-helix contentincreases as the ratio of [θ]_(220 nm)/[θ]_(208 nm) is about 1,confirming the formation of coiled-coil structures between peptide-E andpeptide-K.

FIG. 10 shows a, Transmission electron microscopy image (TEM) of theDPPC liposomes (scale bar, 100 nm); b, Size distribution histogram ofthe liposomes shown in panel a; cryogenic scanning electron microscopy(c) and TEM (d) of the interdigitated bilayer sheets made from the DPPCliposomes (c scale bar, 2 µm; d scale bar, 100 nm).

FIG. 11 shows (a) Schematic illustration and (b) reaction equations ofthe enzymatic reactions involving glucose oxidation catalyzed by glucoseoxidase (GOx) and hydrogen peroxide breakdown by catalase (CAT). Thedecomposition of the undesired hydrogen peroxide can regenerate oxygento facilitate glucose oxidation.

FIG. 12 is a representative cryogenic transmission electron microscopy(cryoTEM) image of the final vesicles-in-vesicle superstructures. Scalebar: 100 nm.

FIG. 13 shows (a) SDS-PAGE analysis of the purified glucose transporter2 (GLUT2). (b) Uncropped western blots for FIG. 1 panel f. Lanes usedfor FIG. 1 panel f are indicated by a rectangle, where Lane 1, 2 and 3respectively represent the blots of GLUT2, liposomal superstructuresinserted with GLUT2, and pure liposomal superstructures. The lanes onthe left side of lane 1 and right side of lane 3 are GLUT2 isolated fromdifferent batches of bacteria.

FIG. 14 shows a, Unionized and ionized forms of HPTS (pK_(a) = 7.2). b,pH titrations of the relative fluorescence intensities of HPTS excitedat 406 nm and 460 nm (HEPES buffer, 5 mM, 100 mM NaCl). The fluorescenceintensities of HPTS at 514 nm excited by 406 (I₄₀₆) and 460 (I₄₆₀) nmwere strongly dependent on the degree of ionization of the 8-hydroxylgroup and hence on the medium pH. Data points represent mean ± SD (n =3).

FIG. 15 is a set of graphs depicting pH variation inside AβCs at modestglucose concentrations with different gramicidin insertion in (a) 300mg/dL and (b) 200 mg/dL glucose solutions. The ratio in the figure labelindicates the ratio of gramicidin-to-dipalmitoylphosphatidylcholinelipid. c, d, Reversible pH variation as a result of alternativelyswitching environmental glucose concentrations. Data points representmean ± SD (n = 3).

FIG. 16 is a set of graphs depicting pH changes inside control AβCs withno glucose transporter 2 reconstitution in (a) 400 md/dL and (b) 100mg/dL glucose solutions, and glucose transporter 2inhibitor-Cytochalasin B pre-treated AβCs in (c) 400 md/dL and (d) 100mg/dL glucose solutions. The ratios in the figure represent the molarratio of the added GA-to-lipid of the outer large liposomal vesicles. Inall the control groups, no significant pH variation was observedcompared to their counterparts in the experimental group over the sametime. The slower decrease in pH is likely induced by the passivediffusion of glucose into the control AβCs. These results show glucosetransporter 2 was responsible for the uptake of glucose into AβC, inwhich subsequent glucose oxidation induced pH variation. Data pointsrepresent mean ± SD (n = 3).

FIG. 17 shows a, FRET assay to study the dehybridization of thepH-sensitive DNA duplex which bridges the PEG shield and the ISVsurface, accompanying the glucose metabolism by AβCs in 300 mg/mL and200 mg/mL glucose solutions. F₀ and F_(t) represent the fluorescenceintensity measured before and at time t after addition into glucosesolutions. b and c, Reversible quenching and recovery of thefluorescence of DNA-donor to demonstrate the reversible attachment anddetachment of the PEG shield at high and low glucose solutions. Datapoints represent mean ± SD (n = 3).

FIG. 18 is a graph depicting a FRET assay to study the dehybridizationof the non-pH-sensitive DNA duplex which bridges the PEG shield and theISV surface, accompanying the glucose metabolism by AβCs in 400 mg/mLand 100 mg/mL glucose solutions. F₀ and F_(t) represent the fluorescenceintensity measured before and at time t after addition into glucosesolutions. The results demonstrated that no disassociation of thecontrol duplex DNA occurred at either high or low glucose levels due tothe lack of pH-sensitive properties. The fluorophore and quenchermodification information was shown in Table 1. Data points representmean ± SD (n = 3).

FIG. 19 shows a, FRET assay to study the interactions of peptide-E andpeptide-K at modest hyperglycemic concentrations. F₀ and F_(t) representthe fluorescence intensity measured before and at time t after additioninto glucose solutions. b and c, Step decrease in fluorescence intensityof peptide-donor by switching the glucose concentrations. Data pointsrepresent mean ± SD (n = 3).

FIG. 20 is a graph depicting a FRET assay to study the interactions ofthe peptide anchored on OLVs and ISVs surfaces at different glucoseconcentrations, where the ISVs were modified with non-pH-responsive DNAbridged PEG shield, and peptide-K and peptide-E were modifiednitrobenzofuran (peptide-donor) and tetramethylrhodamine(peptide-acceptor), respectively. F₀ and F_(t) represent thefluorescence intensity of peptide-donor measured before and at time tafter addition into glucose solutions. The data indicated that by usingnon-pH-controllable PEG shield, the peptide-K on the ISV surfaces werehindered to interact with the peptide-E on OLV inner surfaces. Datapoints represent mean ± SD (n = 3).

FIG. 21 shows a, Kinetics profiles of lipid mixing betweenlipid-donor/acceptor-labeled ISV and OLV in 300 mg/dL and 200 mg/dlglucose solutions as indicated by the increase in lipid-donor emission.b and c, Step increase in the fluorescence of lipid-donor afteralternatively changing the glucose concentrations. Data points representmean ± SD (n = 3).

FIG. 22 is a graph depicting lipid mixing between ISV and OLV asindicated by the variation in NBD florescence in control AβCs in whichpeptide-K (PK), or peptide-E (PE), or peptide-E and peptide-K (PE/PK)were omitted. Lacking the machinery for membrane fusion, no lipid mixingwas detected at either high or low glucose concentrations. Data pointsrepresent mean ± SD (n = 3).

FIG. 23 shows a, Fluorescence spectra of lipid-donor/lipid-acceptorco-labeled ISV before (red line) and after (green line) reduction bysodium dithionate. b, Kinetics profiles showed the mixing of theinner-layer lipids of lipid-donor/lipid-acceptor co-labeled ISV with OLVin 400 mg/dL and 100 mg/dl glucose solutions as indicated by theincrease in lipid-donor emission. Data points represent mean ± SD (n =3).

FIG. 24 is a graph depicting an insulin standard curve obtained byCoomassie Plus protein assay. Data points represent mean ± SD (n = 3).

FIG. 25 shows a, In vitro accumulated insulin release from AβCsincubated in solutions with modest hyperglycemic concentrations. Datapoints represent mean ± SD (n = 3). b, 2.5 D confocal laser microscopyimages showed the fluorescence intensity and distribution of fluoresceinlabeled-insulin from AβCs before and after incubation in solutionscontaining different concentrations of glucose.

FIG. 26 is a set of graphs depicting in vitro accumulated insulinrelease from control AβCs (a) lacking glucose sensing machinery (glucosetransporter 2, glucose oxidase/catalase, and gramicidin) and (b) with nomembrane fusion machinery (peptide-E and peptide-K) at different glucoselevels. c and d respectively showed the rate of the pulsatile insulinrelease profile by the control AβCs used in a and b in response toglucose concentration switch between 400 and 100 mg/dL. Data pointsrepresent mean ± SD (n = 3).

FIG. 27 is a graph depicting in vitro accumulated insulin release fromAβCs in mild acidic environment. Data points represent mean ± SD (n =3).

FIG. 28 is a graph depicting the CD spectra of the solutions containingnative insulins and released insulins from AβCs incubated with 400 mg/dLglucose. Overall secondary structure of released insulins was maintainedto that of native insulins.

FIG. 29 is a graph comparing the bioactivity of the insulin ‘secreted’by AβCs after incubation with 400 mg/dL glucose and that of nativeinsulin. Results showed that the bioactivity of insulin was highlyretained during the AβC preparation and release test. Data pointsrepresent mean ± SD (n = 5).

FIG. 30 is a set of photographs depicting AβC integratedthermoresponsive PF127 (40 wt%) solution immediately formed a hydrogelat 37° C. in vitro (a), and 1 min after subcutaneously injection (b). Asseen in b, the hydrogel presented as a ‘bump’ on the dorsum of themouse.

FIG. 31 shows representative cryogenic scanning electron microscopy(cryoSEM) images of the AβC integrated thermoresponsive PF127 solutionshown in FIG. 30 . Scale bars: 1 µm.

FIG. 32 is a graph depicting in vivo intraperitoneal glucose tolerancetest (IPGTT) performed toward diabetic mice on day five following AβCtreatment in comparison to the healthy control mice. Data pointsrepresent mean ± SD (n = 5).

FIG. 33 shows (a) In vivo intraperitoneal glucose tolerance test (IPGTT)performed toward diabetic mice at 24, 36, and 48 h following AβCtreatment in comparison to the healthy control mice. Data pointsrepresent mean ± SD (n = 5). (b) IPGTT performed toward diabetic mice at24, 36, and 48 h following treatment with PBS (Blank) and control AβCs.The remained high blood glucose levels in the control groups as well asthe insignificant difference in blood glucose variation betweenPBS-treated and control AβC-treated groups demonstrated the lack ofresponsiveness of the control AβCs. Data points represent mean ± SD (n =5).

FIG. 34 is a bar chart depicting in vivo intraperitoneal glucosetolerance test (IPGTT) performed towards wild-type mice transplantedwith AβCs that were loaded with human serum albumin (HSA) as a reporterprotein. The human serum albumin in the serum of each group weremeasured by ELISA at 2 h after intraperitoneal injection of 50 mgglucose/mouse. Data points represent mean ± SD (n = 5).

FIG. 35 is a bar chart depicting results of a cytotoxicity assay ofinsulin-free AβC toward HeLa cells after 24 h incubation. Data pointsrepresent mean ± SD (n = 3).

FIG. 36 shows hematoxylin and eosin-stained sections of subcutaneouslyinjected with PBS solution (blank), PF127 thermogel, or ‘transplanted’with AβC, AβC_((no) _(insulin)), AβC_((no) _(PE/PK)), or AβC_((no)_(GSM)) after four weeks, respectively. Results showed that all thecomponents were degraded and no noticeable inflammatory region orfibrotic encapsulation was observed, indicating the biocompatibility ofthe artificial assemblies. Scale bar: 150 µm.

FIG. 37 is a graph depicting the average body weight of the diabetesmice after injection with PBS solution (blank), PF127 thermogel, or‘transplanted’ with AβC, AβC_((no) _(insulin)), AβC_((no) _(PE/PK)), orAβC_((no) _(GSM)) for four weeks, respectively. Data points representmean ± SD (n = 5).

FIG. 38 is a set of bar charts depicting serum levels of TNF-α, IL-1βand IL-6 in diabetes mice after injection with PBS solution (blank),PF127 thermogel, or ‘transplanted’ with AβC, AβC_((no) _(insulin)),AβC_((no) _(PE/PK)), or AβC_((no) _(GSM)), respectively. Data pointsrepresent mean ± SD (n = 5).

DETAILED DESCRIPTION

Pancreatic β-cells precisely sense blood glucose fluctuations and inturn secret insulin to maintain normoglycemia. Disclosed herein is anovel particle having a sense-and-response system for the delivery of atherapeutic agent (e.g., insulin), which can be useful as an artificialβ-cell. This system is the first of its kind to sense glucose levels andreadily secrete insulin using a vesicle fusion-mediated behavior. Theparticle contains a “vesicle-in-vesicle” superstructure that, in someembodiments, is spatially equipped with a pH-altering agent (e.g., aglucose metabolism system) and membrane fusion machinery. For instance,an inner small liposomal vesicle (ISV) can be loaded with insulin tomimic the storage granules inside mature β-cells, while an outer largevesicle (OLV) can mimic a plasma membrane. Metabolism of glucose withinthe particle can be sensed (e.g., by a glucose-responsive component) andtied to delivery of a therapeutic agent via membrane fusion. Changes inpH within the particle due to glucose metabolism facilitate membranefusion. The inventors discovered that the particle can effectivelydistinguish between high and normal glucose levels and respondappropriately with the release of entrapped therapeutic agent. Thus, theparticle has the capability to mimic functions of pancreatic β-cells bysensing and distinguishing hyperglycemic and normoglycemic conditionsvia metabolizing glucose, and responding to hyperglycemic conditions byreleasing a therapeutic agent though an exocytosis-like membrane fusionmechanism. At least one of several goals achieved by the particle isdelivery of a therapeutic agent (e.g. insulin) in physiologic conditionsin which presence of the therapeutic agent would be beneficial (e.g.hyperglycemia), and avoiding the delivery of a therapeutic agent inphysiologic conditions in which the presence of the therapeutic agentwould provide little or no therapeutic advantage or, in some instances,even cause deleterious effects (e.g. normoglycemia or hypoglycemia).

As depicted in an example embodiment in panel a of FIG. 1 , glucose canbe taken up by an anchored glucose transporter 2 and subsequentlyoxidized into gluconic acid by glucose oxidase. The released protons canrapidly decrease the pH in the system. The net pH variation can bebalanced by efflux through proton channels-gramicidin A inserted in theOLV membrane, which facilitates more significant variation in theinternal pH levels in hyperglycemic conditions. Subsequently, the low pHassociated with high glucose concentrations can trigger dehybridizationof the PEG5000-conjugated cytosine-rich DNA (PEG-CDNA) and thecholesterol-ended guanine-rich DNA (GDNA-CH) anchored on the ISV, whichsterically deshields peptide-K, making it available to form coiled coilswith peptide-E (Marsden, H. et al., Chem. Soc. Rev. 40, 1572-1585(2011); Lygina, A. et al., Angew. Chem. Int. Ed. 50, 8597-8601 (2011);Robson Marsden, H., et al., Chem. Int. Ed. 48, 2330-2333 (2009);Meyenberg, K., et al., Chem. Commun. 47, 9405-9407 (2011); Tomatsu, I.et al., J. Mater. Chem. 21, 18927-18933 (2011); Kong, L., et al., Angew.Chem. Int. Ed. 55, 1396-1400 (2016); Gong, Y., et al., J. Am. Chem. Soc.130, 6196-6205 (2008); Chan, Y. et al., Proc. Natl Acad. Sci. USA 106,979-984 (2009); Steinmetz, M. et al., Proc. Natl Acad. Sci. USA 104,7062-7067 (2007)) on the inner surface of OLV. Zippering up of the twopeptides pulls the membranes of ISV and OLV tightly together and forcesfusion, upon which insulin is “exocytosed”. When glucose concentrationdeclines to a normoglycemic range, glucose uptake can decrease and theinner pH level can increase, which can facilitate the ISVs to re-anchorwith PEG-CDNA and subsequently inhibit the fusion event. In thisbiomimetic manner, the AβC can effectively respond to hyperglycemia andresume the basic low level insulin release under resting conditions.

Reference will now be made in detail to the embodiments of theinvention, examples of which are illustrated in the drawings and theexamples. The present disclosure may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. The following definitions areprovided for the full understanding of terms used in this specification.

Terminology

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an agent” includes a plurality ofagents, including mixtures thereof.

As used herein, the terms “may,” “optionally,” and “may optionally” areused interchangeably and are meant to include cases in which thecondition occurs as well as cases in which the condition does not occur.Thus, for example, the statement that a formulation “may include anexcipient” is meant to include cases in which the formulation includesan excipient as well as cases in which the formulation does not includean excipient.

“Administration” to a subject includes any route of introducing ordelivering to a subject an agent. Administration can be carried out byany suitable route, including oral, topical, intravenous, subcutaneous,transcutaneous, transdermal, intramuscular, intra-joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, byinhalation, via an implanted reservoir, parenteral (e.g., subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional,and intracranial injections or infusion techniques), and the like.“Concurrent administration”, “administration in combination”,“simultaneous administration” or “administered simultaneously” as usedherein, means that the compounds are administered at the same point intime or essentially immediately following one another. In the lattercase, the two compounds are administered at times sufficiently closethat the results observed are indistinguishable from those achieved whenthe compounds are administered at the same point in time. “Systemicadministration” refers to the introducing or delivering to a subject anagent via a route which introduces or delivers the agent to extensiveareas of the subject’s body (e.g. greater than 50% of the body), forexample through entrance into the circulatory or lymph systems. Bycontrast, “local administration” refers to the introducing or deliveryto a subject an agent via a route which introduces or delivers the agentto the area or area immediately adjacent to the point of administrationand does not introduce the agent systemically in a therapeuticallysignificant amount. For example, locally administered agents are easilydetectable in the local vicinity of the point of administration, but areundetectable or detectable at negligible amounts in distal parts of thesubject’s body. Administration includes self-administration and theadministration by another.

“Biocompatible” generally refers to a material and any metabolites ordegradation products thereof that are generally non-toxic to therecipient and do not cause significant adverse effects to the subject.

“Comprising” is intended to mean that the compositions, methods, etc.include the recited elements, but do not exclude others. “Consistingessentially of” when used to define compositions and methods, shall meanincluding the recited elements, but excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

A “control” is an alternative subject or sample used in an experimentfor comparison purposes. A control can be “positive” or “negative.”

“Controlled release” or “sustained release” refers to release of anagent from a given dosage form in a controlled fashion in order toachieve the desired pharmacokinetic profile in vivo. An aspect of“controlled release” agent delivery is the ability to manipulate theformulation and/or dosage form in order to establish the desiredkinetics of agent release.

“Peptide,” “protein,” and “polypeptide” are used interchangeably torefer to a natural or synthetic molecule comprising two or more aminoacids linked by the carboxyl group of one amino acid to the alpha aminogroup of another.

“Pharmaceutically acceptable” component can refer to a component that isnot biologically or otherwise undesirable, e.g., the component may beincorporated into a pharmaceutical formulation of the invention andadministered to a subject as described herein without causingsignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the formulationin which it is contained. When used in reference to administration to ahuman, the term generally implies the component has met the requiredstandards of toxicological and manufacturing testing or that it isincluded on the Inactive Ingredient Guide prepared by the U.S. Food andDrug Administration.

“Pharmaceutically acceptable carrier” (sometimes referred to as a“carrier”) means a carrier or excipient that is useful in preparing apharmaceutical or therapeutic composition that is generally safe andnon-toxic, and includes a carrier that is acceptable for veterinaryand/or human pharmaceutical or therapeutic use. The terms “carrier” or“pharmaceutically acceptable carrier” can include, but are not limitedto, phosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.As used herein, the term “carrier” encompasses, but is not limited to,any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer,lipid, stabilizer, preservatives (e.g., Thimerosal, benzyl alcohol,parabens), binders, fillers, disintegrants, sorbents, solvents, pHmodifying agents, antioxidants, antinfective agents, suspending agents,wetting agents, viscosity modifiers, tonicity agents, and othercomponents and combinations thereof or other material well known in theart for use in pharmaceutical formulations and as described furtherherein. Suitable pharmaceutically acceptable excipients are preferablyselected from materials which are generally recognized as safe (GRAS),and may be administered to an individual without causing undesirablebiological side effects or unwanted interactions. Suitable excipientsand their formulations are described in Remington’s PharmaceuticalSciences, 16th ed. 1980, Mack Publishing Co.

“Pharmacologically active” (or simply “active”), as in a“pharmacologically active” derivative or analog, can refer to aderivative or analog (e.g., a salt, ester, amide, conjugate, metabolite,isomer, fragment, etc.) having the same type of pharmacological activityas the parent compound and approximately equivalent in degree.

“Primer” or “DNA primer” is a short polynucleotide, generally with afree 3'-OH group that binds to a target or “template” potentiallypresent in a sample of interest by hybridizing with the target, andthereafter promoting polymerization of a polynucleotide complementary tothe target. A “polymerase chain reaction” (“PCR”) is a reaction in whichreplicate copies are made of a target polynucleotide using a “pair ofprimers” or a “set of primers” consisting of an “upstream” and a“downstream” primer, and a catalyst of polymerization, such as a DNApolymerase, and typically a thermally-stable polymerase enzyme. Methodsfor PCR are well known in the art, and taught, for example in “PCR: APRACTICAL APPROACH” (M. MacPherson et al., IRL Press at OxfordUniversity Press (1991)). All processes of producing replicate copies ofa polynucleotide, such as PCR or gene cloning, are collectively referredto herein as “replication.” A primer can also be used as a probe inhybridization reactions, such as Southern or Northern blot analyses.Sambrook et al., supra.

“Therapeutic agent” refers to any composition that has a beneficialbiological effect. Beneficial biological effects include boththerapeutic effects, e.g., treatment of a disorder or other undesirablephysiological condition, and prophylactic effects, e.g., prevention of adisorder or other undesirable physiological condition (e.g., Type 1diabetes). The terms also encompass pharmaceutically acceptable,pharmacologically active derivatives of beneficial agents specificallymentioned herein, including, but not limited to, salts, esters, amides,proagents, active metabolites, isomers, fragments, analogs, and thelike. When the terms “therapeutic agent” is used, then, or when aparticular agent is specifically identified, it is to be understood thatthe term includes the agent per se as well as pharmaceuticallyacceptable, pharmacologically active salts, esters, amides, proagents,conjugates, active metabolites, isomers, fragments, analogs, etc.

“Therapeutically effective amount” or “therapeutically effective dose”of a composition (e.g. a composition comprising an agent) refers to anamount that is effective to achieve a desired therapeutic result. Insome embodiments, a desired therapeutic result is the control of type Idiabetes. In some embodiments, a desired therapeutic result is thecontrol of obesity. Therapeutically effective amounts of a giventherapeutic agent will typically vary with respect to factors such asthe type and severity of the disorder or disease being treated and theage, gender, and weight of the subject. The term can also refer to anamount of a therapeutic agent, or a rate of delivery of a therapeuticagent (e.g., amount over time), effective to facilitate a desiredtherapeutic effect, such as pain relief. The precise desired therapeuticeffect will vary according to the condition to be treated, the toleranceof the subject, the agent and/or agent formulation to be administered(e.g., the potency of the therapeutic agent, the concentration of agentin the formulation, and the like), and a variety of other factors thatare appreciated by those of ordinary skill in the art. In someinstances, a desired biological or medical response is achievedfollowing administration of multiple dosages of the composition to thesubject over a period of days, weeks, or years.

“Treat,” “treating,” “treatment,” and grammatical variations thereof asused herein, include the administration of a composition with the intentor purpose of partially or completely preventing, delaying, curing,healing, alleviating, relieving, altering, remedying, ameliorating,improving, stabilizing, mitigating, and/or reducing the intensity orfrequency of one or more a diseases or conditions, a symptom of adisease or condition, or an underlying cause of a disease or condition.Treatments according to the invention may be applied preventively,prophylactically, pallatively or remedially. Prophylactic treatments areadministered to a subject prior to onset (e.g., before obvious signs ofdiabetes), during early onset (e.g., upon initial signs and symptoms ofdiabetes), or after an established development of diabetes. Prophylacticadministration can occur for day(s) to years prior to the manifestationof symptoms of an infection.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value” 10” is disclosed, then “about 10” is alsodisclosed.

Publications cited herein are hereby specifically incorporated byreference in their entireties and at least for the material for whichthey are cited.

Particles

In one aspect, disclosed herein is a particle comprising: an innerliposomal vesicle (ILV) encapsulating a therapeutic agent; an outerliposomal vesicle (OLV) encapsulating the ILV; a membranefusion-promoting agent; and a pH-altering agent.

In some instances, the ILV can be referred to as an insulin-loadedfusogenic small liposomal vesicle, an inner small liposomal vesicle, orISV. In some instances, the OLV can be referred to as an outer largevesicle, or an outer large liposomal vesicle.

The particle contains a vesicle-in-vesicle superstructure in which theILV is encapsulated within the OLV. Because the particle contains asense-and-response system and a vesicle-in-vesicle superstructure, theparticle mimics a biological cell, particularly a pancreatic β-cell. Assuch, the particle is sometimes referred to herein as an artificialβ-cell (AβC).

Typically, ILVs can first be formed, then added to lipid sheets that areinduced to close around an ILV to form the vesicle-in-vesiclesuperstructure. In some embodiments, the OLV encapsulates one or moreILVs. In some embodiments, the OLV encapsulates a plurality of ILVs.Optionally, the OLV encapsulates a mixture of ILVs. Optionally, themixture of ILVs comprises a first ILV encapsulating a first therapeuticagent and a second ILV encapsulating a second therapeutic agent.

Optionally, the OLV is a vesicle comprising lipids. Optionally, the OLVis a liposomal vesicle comprising a lipid membrane such as alipid-bilayer membrane. Lipids used to form the bilayer membrane of theOLV can be any lipids suitable for liposome formation. The lipids can bebiological lipids, for instance lipids extracted from or extractablefrom a biological cell. Optionally, the lipids are phospholipids (e.g.,phosphatidylcholine, phosphatidylethanolamine, etc.). Mixtures orcombinations of lipids can be used to form liposomal lipid bilayermembranes.

The OLV can have a range of sizes, but must be larger than the ILV toencapsulate the ILV. In some embodiments, the OLV can have an averagediameter of greater than 100 nm, 250 nm, 500 nm, 750 nm, 1 µm, 1.5 µm,2.0 µm, 2.5 µm, 3.0 µm, 3.5 µm, 4.0 µm, 4.5 µm, or greater than 5.0 µm.In some embodiments, the OLV can have an average diameter of less than10 µm, 9 µm, 8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or less than 1µm. It is understood that the OLV can have an average diameter rangingfrom any of the minimum values to any of the maximum values describedabove. For example, the OLV can have an average diameter ranging from100 nm to 10 µm, or from 1 µm to 5 µm, etc.

Optionally, the ILV is a vesicle comprising lipids. Optionally, the ILVis a liposomal vesicle comprising a lipid membrane such as alipid-bilayer membrane. Lipids used to form the bilayer membrane of theILV can be any lipids suitable for liposome formation. The lipids can bebiological lipids, for instance lipids extracted from or extractablefrom a biological cell. Optionally, the lipids are phospholipids (e.g.,phosphatidylcholine, phosphatidylethanolamine, etc.) or derivatives ofphospholipids (e.g., egg phosphatidylcholine,dioleoyl-glycero-phosphoethanolamine, palmitoylphosphatidylcholine,etc.). Mixtures or combinations of lipids can be used to form liposomallipid bilayer membranes. The ILV can contain the same or differentlipids compared to the OLV. Typically, selected ILV lipids can befusogenic with the OLV; in other words, the selected ILV lipids can becapable of forming a lipid bilayer membrane that can fuse with the lipidbilayer membrane of the OLV.

The ILV can be a range of sizes, but must be smaller than the OLV to beencapsulated within the OLV. In some embodiments, the ILV can have anaverage diameter of greater than 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm,500 nm, 1 µm, 1.5 µm, 2.0 µm, 2.5 µm, or greater than 3.0 µm. In someembodiments, the ILV can have an average diameter of less than 5 µm, 4µm, 3 µm, 2 µm, 1 µm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm,150 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm,or less than 10 nm. It is understood that the ILV can have an averagediameter ranging from any of the minimum values to any of the maximumvalues described above. For example, the ILV can have an averagediameter ranging from 1 nm to 5 µm, or from 10 nm to 1 µm, or from 10 nmto 100 nm, etc.

The particle comprises an ILV encapsulating a therapeutic agent. Thetherapeutic agent can be any agent capable of being encapsulated within,and subsequently released from, an ILV to impart a therapeutic effect.The therapeutic agent can be encapsulated within the aqueous-filledinterior region of the ILV. For instance, the therapeutic agent can be ahydrophilic compound or biomolecule. The therapeutic agent can bepartially or fully embedded in the lipid bilayer membrane of the ILV.For instance, the therapeutic agent can be a hydrophobic or lipophiliccompound. In some embodiments, the therapeutic agent can be attached tothe ILV, for instance by a linking molecule such as a polyethyleneglycol (PEG) molecule. Optionally, the ILV can encapsulate one or moretherapeutic agents.

In some embodiments, the therapeutic agent is an agent which treatsglycemic imbalance. In some embodiments, the therapeutic agent is anagent which treats hyperglycemia. In some embodiments, the therapeuticagent is an agent which treats diabetes. In some embodiments, thetherapeutic agent can be a peptide, protein, signally molecule (e.g.,hormone), small molecule, carbohydrate, nucleic acid molecule, lipid,organic molecule, biologically active inorganic molecule, orcombinations thereof. Optionally, the therapeutic agent can compriseinsulin or a biologically active compound derived from insulin.

In some embodiments, the therapeutic agent can be used to treatdiabetes, such as insulin, alpha-glucosidase inhibitors (e.g., acarbose,miglitol), biguanides (e.g., metformin), dopamine agonists (e.g.,bromocriptine), DPP-4 inhibitors (e.g., alogliptin,alogliptin-metformin, alogliptin-pioglitazone, linagliptin,linagliptin-empagliflozin, linagliptin-metformin, saxagliptin,saxagliptin-metformin, sitagliptin, sitagliptin-metformin, sitagliptinand simvastatin), glucagon-like peptides/incretin mimetics (e.g.,albiglutide, dulaglutide, exenatide, exenatide extended-release,liraglutide, semaglutide), meglitinides (e.g., nateglinide, repaglinide,repaglinide-metformin), sodium glucose transporter (SGLT) 2 inhibitors(e.g., dapagliflozin, dapagliflozin-metformin, canagliflozin,canagliflozin-metformin, empagliflozin, empagliflozin-linagliptin,empagliflozin-metformin, ertugliflozin), sulfonylureas (e.g.,glimepiride, glimepiride-pioglitazone, glimepiride-rosiglitazone,gliclazide, glipizide, glipizide-metformin, glyburide,glyburide-metformin, chlorpropamide, tolazamide, tolbutamide),thiazolidinediones (e.g., rosiglitazone, rosiglitazone-glimepiride,rosiglitazone-metformin, pioglitazone, pioglitazone-alogliptin,pioglitazone-glimepiride, pioglitazone-metformin), and combinationsthereof.

Optionally, the ILV can encapsulate an additional agent or,alternatively or in addition to, the OLV can encapsulate a first ILVencapsulating a therapeutic agent and a second ILV encapsulating anadditional agent. Optionally, the additional agent can be a therapeutic,prophylactic, or diagnostic agent.

The herein disclosed particle comprises a membrane fusion-promotingagent. The membrane fusion-promoting agent can promote fusion betweenthe ILV and the OLV or, in some embodiments, between an ILV lipidbilayer membrane and an OLV lipid bilayer membrane. Optionally, themembrane fusion-promoting agent is attached to the surface of a lipidbilayer membrane. Such attachment can be noncovalent (e.g., byhydrophobic insertion) or covalent (e.g., by covalent linkage directlyto a lipid or to a linking molecule). When accessible, the membranefusion-promoting agent can be specifically bound and subsequentlypromote membrane fusion. Membrane fusion activity can be inhibited by,for example, blocking accessibility of the membrane fusion-promotingagent or by altering the conformation of the membrane fusion-promotingagent. In some embodiments, the membrane fusion-promoting agent cancycle between an accessible state and a blocked state.

In some embodiments, the membrane fusion-promoting agent can bespecifically bound by dimerizing. For example, a first copy of themembrane fusion-promoting agent can specifically bind a second copy ofthe membrane fusion-promoting agent. Specific binding of the membranefusion-promoting agent can facilitate fusion between two or moremembranes (e.g. lipid bilayer membranes). Without being limited to aparticular mechanism, the specific binding of the membranefusion-promoting agent can bring two or more membranes to within closeproximity, particularly when the membrane fusion-promoting agent isbound to the surface of a membrane to be fused, and facilitate locallipid rearrangements which results in fusing of the membranes. As usedherein, the term “specific binding” and grammatical variations thereofrefers to the binding of a first component to a second component with anaffinity greater than the affinity of the first component for generallyany other component in the particle (e.g., background or non-specificbinding). In some embodiments, the term “specific binding” refers to thebinding of two or more components to form a biological complex having abiological function, wherein the two or more components have an affinityfor each other greater than the affinity for non-specific bindingcomponents.

Optionally, the membrane fusion-promoting agent comprises a firstmembrane fusion-promoting agent and a second membrane fusion-promotingagent. Optionally, the first membrane fusion-promoting agent is attachedto the ILV, for instance to the outer surface of the ILV, and the secondmembrane fusion-promoting agent is attached to the OLV, for instance tothe inner surface of the OLV. Optionally, the attachment of membranefusion-promoting agents to lipid vesicles is reversed: the firstmembrane fusion-promoting agent is attached to the OLV, for instance tothe inner surface of the OLV, and the second membrane fusion-promotingagent is attached to the ILV, for instance to the outer surface of theILV.

The membrane fusion-promoting agent can be any suitable agent which,when specifically bound, facilitates membrane fusion. Thus, the membranefusion-promoting agent can be any protein, peptide, nucleic acid, lipid,carbohydrate, synthetic polymer, small molecule attached to e.g., alipid or synthetic polymer (e.g., a polyethylene glycol (PEG) orpegylated-molecule), etc. In some embodiments, the membranefusion-promoting agent comprises a biological molecule. In someembodiments, the membrane fusion-promoting agent can facilitate membranefusion by positioning (e.g., by pulling) the OLV and ILV in sufficientlyclose proximity for membrane fusion to occur.

In some embodiments, the membrane fusion-promoting agent comprises aSoluble NSF Attachment Protein Receptor (SNARE) protein. Optionally, themembrane fusion-promoting agent is selected from Peptide-K, Peptide-E,or combinations thereof. Peptide-K refers to a peptide comprising theamino acid sequence (KIAALKE)₃ (SEQ ID NO: 1). Peptide-E refers to apeptide comprising the amino acid sequence (EIAALEK)₃ (SEQ ID NO:2). Insome embodiments, the membrane fusion-promoting agent comprises a firstmembrane fusion-promoting agent and a second membrane fusion-promotingagent, in which the first membrane fusion-promoting agent is a firstSNARE polypeptide (e.g., Peptide-K) and the second membranefusion-promoting agent is a second SNARE polypeptide (e.g., Peptide-E).When attached and accessible on the outer surface of an ILV, a firstSNARE polypeptide (e.g., Peptide-K) can specifically bind a second SNAREpolypeptide (e.g., Peptide-E) that is attached and accessible on theouter surface of an OLV. Specific binding of Peptide-K and Peptide-E canthus result in membrane fusion between the ILV and OLV.

In some embodiments, the first and second membrane fusion-promotingagents can be complementary nucleic acid molecules (e.g., a firstsingle-stranded DNA molecule which binds a complementary secondsingle-stranded DNA molecule). In some embodiments, the first and secondmembrane fusion-promoting agents can be a combination of molecule types.For example, a DNA-binding protein can specifically bind adouble-stranded DNA molecule, or a lipid-binding protein canspecifically bind a lipid integrated in a membrane to be fused.

The herein disclosed particle comprises a pH-altering agent. ThepH-altering agent can reduce the pH inside the OLV. A particularlypreferable pH-altering agent is an agent which alters the pH in responseto a stimulus, for example a stimulus presenting information aboutconditions outside the OLV. For example, the pH-altering agent can alterthe pH in response to biomolecule or substrate concentrations in thesurrounding medium (e.g., blood-glucose concentrations). In someembodiments, the pH-altering agent can reduce the pH.

In some embodiments, the pH-altering agent comprises a biologicalmolecule. In some embodiments, the pH-altering agent comprises apolypeptide, for instance an enzyme. Optionally, the pH-altering agentcomprises a glucose-responsive enzyme. In some embodiments, thepH-altering agent comprises glucose oxidase (GOx). In such anembodiment, GOx metabolizes glucose within the OLV, releasing protonsand reducing the pH within the OLV. Thus, as the glucose concentrationwithin the OLV increases, GOx has access to increased amounts of glucosesubstrate, resulting in concomitant pH reduction.

In some embodiments, the particle can further contain an enzyme forscavenging metabolism-byproducts, for example a peroxide-metabolizingenzyme. For example, GOx-mediated glucose metabolism may result inproduction of peroxides, particularly hydrogen peroxide. An enzyme canbe present within the OLV which can degrade or scavenge hydrogenperoxide byproduct. In some embodiments, the peroxide-metabolizingenzyme can be catalase (CAT). Catalase provides a further benefit toGOx-mediated glucose metabolism in that catalase can regenerate oxygento facilitate further glucose oxidation.

In some embodiments, the particle can further contain a small moleculetransporter. A small molecule transporter can facilitate the exchange ofsmall molecules between the OLV and the exterior medium in which theparticle resides. In some embodiments, the small molecule transporter isintegrated in the OLV lipid bilayer liposomal membrane. In someembodiments, the small molecule transporter can be a component of alarger membrane-associated transport system (e.g., a lipid-coupled,multi-protein membrane transport system). In some embodiments, the smallmolecule transporter is a transmembrane transport protein. The smallmolecule transporter need not be a dedicated transporter; the smallmolecule transporter can transport an array of substrates. Preferably,the small molecule transporter transports a small molecule of interestto which the particle “senses” and responds to. As an example, highglucose conditions (e.g., hyperglycemic levels in the blood) can be astimulus to which the particle responds to. As glucose concentrations inthe medium increase, the small molecule transporter (e.g., a glucosemembrane transporter) can relay this information to within the OLV bytransporting glucose to the interior of the OLV. Thus, hyperglycemicconditions can stimulate increased glucose transport, which can increaseGOx-mediated glucose metabolism, thereby resulting in reduced pH withinthe OLV. In some embodiments, the small molecule transporter comprises aglucose transporter (e.g., Glucose Transporter 2 (GLUT2); also known assolute carrier family 2 or SLC2A2).

In some embodiments, the particle can further contain a protontransporter. In some embodiments, the proton transporter is integratedinto the OLV lipid bilayer liposomal membrane. In some embodiments, theproton transporter comprises a prokaryotic polypeptide, for instance apore-forming protein. Preferably, the proton transporter is oriented inthe membrane for the export of protons. Rapid import and subsequentmetabolism of a substrate (e.g., glucose) that, when metabolizedreleases protons, can result in a rapid decrease in pH. In someembodiments, it can be advantageous to temper pH fluctuations to, forexample, exert greater control over the rapidity of change or the degreeof change. In such embodiments, proton export can counter intravesicleproton production and/or release. In some embodiments, the protontransporter can be Gramicidin A, B, or C.

In some embodiments, the particle can further contain a membranefusion-inhibiting agent. In some aspects, it may be desirable to preventmembrane fusion, for example between the OLV and ILV. Because ILV-OLVfusion results in release of the therapeutic agent encapsulated in theILV in some embodiments, release of the therapeutic agent can optionallybe restricted or controlled by inhibiting ILV-OLV fusion. The membranefusion-inhibiting agent can be any agent suitable to inhibit ILV-OLVfusion (for example, by inhibiting lipid membrane fusion) including, forexample, peptides, proteins, synthetic polymers, nucleic acids, and thelike.

One particularly advantageous membrane fusion-inhibiting agent is onewhich shields access to the membrane fusion-promoting agent, for exampleby steric blockade. Such a mechanism can prevent the membranefusion-promoting agent from being specifically bound, thereby inhibitinga key facilitating step for membrane fusion. Thus, in some embodiments,a membrane fusion-promoting agent containing a first SNARE polypeptide(e.g., Peptide-K) can be blocked from specifically binding a secondSNARE polypeptide (e.g., Peptide-E) by a larger molecule which shieldsaccess to the first SNARE polypeptide, the second SNARE polypeptide, orboth. Thus, in some embodiments, the membrane fusion-inhibiting agentcan be attached to the ILV, the OLV, or both.

In some embodiments, the membrane fusion-inhibiting agent can bemembrane embedded or attached to lipids of a membrane. In someembodiments, the membrane fusion-inhibiting agent contains a moleculelarger than the membrane fusion-promoting agent and thus capable ofshielding the membrane fusion-promoting agent. Optionally, membranefusion-inhibiting agent contains a polyethylene glycol (PEG) molecule.The PEG can be any biocompatible PEG have a sufficiently large size toinhibit specific binding of the membrane fusion-promoting agent. In someembodiments, the PEG has a molecular weight of at least 0.1, 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some embodiments, the PEG can beconjugated with other molecules (e.g., a nucleic acid, lipid,carbohydrate, small molecule, peptide, protein, and the like).

In some embodiments, the membrane fusion-inhibiting agent can also be,for example, a soluble mediator which blocks specific binding of themembrane fusion-promoting agent. As an example, the membranefusion-promoting agent can be a nucleic acid and the membranefusion-inhibiting agent can be a soluble DNA-binding protein.

In some embodiments, the membrane fusion-inhibiting agent can beresponsive to the stimulus the particle can “sense.” Thus, the membranefusion-inhibiting agent can be blocked from inhibiting membrane fusionunder certain conditions. For example, a particle which “senses” highglucose levels in the surrounding medium (e.g., hyperglycemic bloodlevels) by transporting and metabolizing glucose, thereby reducing thepH within the OLV, can further block the activity of the membranefusion-inhibiting agent under hyperglycemic conditions. Because highglucose levels in the medium result in pH reduction within the OLV, themembrane fusion-inhibiting agent can be a pH-responsive agent. Forexample, the membrane fusion-inhibiting agent can be degraded,denatured, sequestered, or otherwise inactivated at low pH. In someembodiments, the membrane fusion-inhibiting agent can be anacid-degradable polyethylene glycol (PEG) molecule. In such embodiments,reduction in pH results in degraded PEG molecules, thereby releasing thesteric blockade of the membrane fusion-promoting agent and facilitatingsubsequent ILV-OLV membrane fusion. A pH-responsive agent (e.g., anacid-degradable PEG) can be responsive at a pH of 7.3 or less, 7.2 orless, 7.1 or less, 7.0 or less, 6.9 or less, 6.8 or less, 6.7 or less,6.6 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 orless, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less,5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, 5.0 or less, 4.9 orless, 4.8 or less, 4.7 or less, 4.6 or less, 4.5 or less, 4.4 or less,4.3 or less, 4.4 or less, 4.3 or less, 4.2 or less, 4.1 or less, or 4.0or less.

Optionally, the particle can be formulated in a medicament. The particlecan be formulated in any suitable medicament including, for example, butnot limited to, an injectable solution, an intravenous drip, a hydrogel,and the like. The medicament can comprise a pharmaceutically acceptableexcipient. In some embodiments, the medicament can further comprise anadditional diagnostic or therapeutic agent.

Methods

Disclosed herein is a method of delivering a therapeutic agent. Themethod can include delivering a therapeutic agent to a subject. Themethod can include providing a particle comprising an inner liposomalvesicle (ILV) encapsulating a therapeutic agent, an outer liposomalvesicle (OLV) encapsulating the ILV, a membrane fusion-promoting agent,and a pH-altering agent. The method can further include triggering ILVand OLV fusion. The method can further include releasing the therapeuticagent outside of the OLV.

The particle can be any particle disclosed herein.

In some embodiments, the triggering step can be in response to astimulus (e.g., solute concentration in the surrounding medium). In someembodiments, the triggering step can be preceded by a chemical orenzymatic reaction, for instance enzymatic release of protons. In someembodiments, the triggering step can be in response to an altered pH(e.g., a reduced pH) within the OLV.

In some embodiments comprising a small molecule transporter, the methodcan include transporting a small molecule (e.g., glucose). In suchembodiments, the transporting step can include transporting a smallmolecule substrate into the OLV via the small molecule transporter. Insome embodiments, the triggering step can be coupled to the transportingstep. For instance, the triggering step can proceed only after thetransporting step proceeds.

In some embodiments, the method can include sensing a small molecule.The sensing step can be any form of molecular interaction. For exampleand without limitation, the sensing step may be performed by binding,degrading, cleaving, sequestering, conjugating, activating,metabolizing, etc. the small molecule. As an example, glucose may bedegraded or metabolized by an enzyme. In some aspects, the sensing stepis performed by the pH-altering agent. In some aspects, the sensing stepcomprises degrading the small molecule by the pH-altering agent.

In some aspects, the sensing step provides a signal to which theparticle responds. In some embodiments, the sensing step reduces the pHin the OLV. The pH can optionally be reduced below the pH of thesurrounding medium. In some embodiments, the pH can be reduced to 7.3 orless, 7.2 or less, 7.1 or less, 7.0 or less, 6.9 or less, 6.8 or less,6.7 or less, 6.6 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 orless, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less,5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, 5.0 orless, 4.9 or less, 4.8 or less, 4.7 or less, 4.6 or less, 4.5 or less,4.4 or less, 4.3 or less, 4.4 or less, 4.3 or less, 4.2 or less, 4.1 orless, or 4.0 or less.

In some embodiments comprising a particle comprising a protontransporter, the method can further comprise regulating pH within theOLV. pH regulation can occur in part via export of protons by the protontransporter. In some embodiments, the pH can be regulated by aproton-sequestering molecule.

In some embodiments comprising a particle comprising a membranefusion-inhibiting agent, the method can further comprise inhibitingmembrane fusion. Membrane fusion can be inhibited under any generalcondition or under one or more specific conditions. In embodimentscomprising an inhibiting step, the triggering step can be blocked frominitiating, continuing, or accelerating. Thus, the inhibiting step cantemporarily stall progression of the method until the inhibiting stepterminates. The inhibiting step can proceed for any amount of time(seconds, minutes, hours, days, weeks, months, etc.). The inhibitingstep can fully or partially inhibit the triggering step.

In embodiments comprising an inhibiting step, the method may furthercomprise a deshielding step. Thus, in some aspects, the method cancomprise deshielding the membrane fusion-promoting agent. The membranefusion-inhibiting agent can inhibit the membrane fusion-promoting agentin any number of ways which blocks promotion of membrane fusionactivity. Thus, “deshielding” is intended to refer to any removal,negation, reduction, etc. of the inhibiting activity of the membranefusion-inhibiting agent on the membrane fusion-promoting agent. Forexample and without limitation, deshielding can refer to degrading,denaturing, sequestering, or otherwise inactivating the membranefusion-inhibiting agent which results in the ability of the membranefusion-promoting agent to be specifically bound. As an example, themethod can comprise deshielding the membrane fusion-promoting agent byacid-degradation of the membrane fusion-inhibiting agent in response todecreased pH within the OLV.

In some embodiments, the inhibiting and deshielding steps can berepeated one or more times, two or more times, three or more times, orfour or more times. In some embodiments, the inhibiting and deshieldingsteps can be cyclical. In some embodiments, repetition of the inhibitingand deshielding steps depends on the sensing step. As an example, thesensing step may reduce the pH in the OLV when the medium contains highlevels of glucose, thereby initiating the deshielding step. When theglucose levels in the medium become reduced, the sensing step can ceasereducing the pH in the OLV, and a net increase in pH within the OLV canoccur. In such instances, the deshielding step can cease, and theinhibiting step can initiate. The number of times the inhibiting anddeshielding steps can be repeated is limited by the number of ILVs inthe OLV which successfully proceed through to the releasing step. Oncean ILV fuses with the OLV and releases encapsulated therapeutic agent,that ILV is no longer available for future releasing steps.

The termination of the deshielding step and initiation of the inhibitingstep can occur by any method which restores the inhibiting activity ofthe membrane fusion-inhibiting agent. For example, protonation anddeprotonation can, in some embodiments, regulate the inhibiting activityof the membrane fusion-inhibiting agent. In some embodiments, themembrane fusion-inhibiting agent can be cleaved or degraded in responseto reduced pH. In some embodiments, the membrane fusion-inhibiting agentcan be reconstituted in response to increased pH, thereby restoring theinhibiting activity of the membrane fusion-inhibiting agent.

The method includes a triggering step comprising triggering ILV and OLVfusion. In some embodiments, the triggering step b) results from thesensing step. The triggering step can include any molecular “trigger”suitable to trigger ILV and OLV fusion. In some embodiments, the sensingstep results in decreasing pH, wherein decreasing pH triggers membranefusion activity. More specifically, in some embodiments, the sensingstep results in decreasing pH, wherein decreasing pH results indeshielding the membrane fusion-promoting agent, wherein deshieldingresults in specific binding of the membrane fusion-promoting agent. Insome embodiments, the triggering step can be facilitated by specificbinding of the membrane fusion-promoting agent. In some embodiments, thetriggering step b) results in fusion of the ILV lipid bilayer liposomalmembrane and the OLV lipid bilayer liposomal membrane.

The method includes releasing the therapeutic agent outside of the OLV.Fusion of the ILV with the OLV results in an “exocytosis-like”phenomenon in which encapsulated therapeutic agent is released to thesurrounding medium outside the OLV. In some aspects, at least a portionof the therapeutic agent is released. In some aspects, all of thetherapeutic agent in an ILV is released. In some aspects, two or moreILVs fuse with the OLV, wherein each ILV releases encapsulatedtherapeutic agent. Therapeutic agent release by multiple ILVs can occurconcurrently, sequentially, or over a relatively long period of time. Inembodiments which include two or more repetitions of inhibiting anddeshielding steps, two or more releasing steps can also proceed. Forexample, the method may include a first inhibiting step, then a firstdeshielding step, then a first releasing step, then a second inhibitingstep, then a second deshielding step, then a second releasing step, andso forth.

In some aspects, the releasing step c) is facilitated in hyperglycemicconditions by, for example, inclusion of a deshielding step. In someaspects, the releasing step c) is inhibited in normoglycemic orhypoglycemic conditions by, for example, inclusion of an inhibitingstep.

The method includes delivery of an agent in a subject. The subject canbe any mammalian subject, for example a human, dog, cow, horse, mouse,rabbit, etc. In some embodiments, the subject comprises a small moleculewhich can be sensed and responded to by the particle. In someembodiments, the subject has a disease or condition. In someembodiments, the subject has hyperglycemia. In some embodiments, thesubject has diabetes.

In another aspect, provided herein is a method for treating a disease ina subject. In some aspects, the subject is in need of treating thedisease. In some aspects, the method includes administering to a subjecta particle comprising an inner liposomal vesicle (ILV) encapsulating atherapeutic agent, an outer liposomal vesicle (OLV) encapsulating theILV, a membrane fusion-promoting agent, and a pH-altering agent.

The particle can be any particle disclosed herein.

In some embodiments, the administering step can include any method ofintroducing the particle into the subject appropriate for the particleformulation. The administering step can include at least one, two,three, four, five, six, seven, eight, nine, or at least ten dosages. Theadministering step can be performed before the subject exhibits diseasesymptoms (e.g., prophylactically), or during or after disease symptomsoccur. The administering step can be performed prior to, concurrentwith, or subsequent to administration of other agents to the subject. Insome embodiments, the administering step is performed withoutadministration of immunosuppressive agents. In some embodiments, theparticle is administered in a hydrogel.

In some embodiments, a subsequent administration is provided at leastone day after a prior administration, or at least two days, at leastthree days, at least four days, at least five days, or at least six daysafter a prior administration. In some embodiments, a subsequentadministration is provided at least one week after a prioradministration, or at least two weeks, at least three weeks, or at leastfour weeks after a prior administration. In some embodiments, asubsequent administration is provided at least one month, at least twomonths, at least three months, at least six months, or at least twelvemonths after a prior administration.

The amount of the disclosed compositions administered to a subject willvary from subject to subject, depending on the nature of the disclosedcompositions and/or formulations, the species, gender, age, weight andgeneral condition of the subject, the mode of administration, and thelike. Effective dosages and schedules for administering the compositionsmay be determined empirically, and making such determinations is withinthe skill in the art. The dosage ranges for the administration of thedisclosed compositions are those large enough to produce the desiredeffect (e.g., to alter insulin levels). The dosage should not be solarge as to outweigh benefits by causing adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like, althoughsome adverse side effects may be expected. The dosage can be adjusted bythe individual clinician in the event of any counterindications.Generally, the disclosed compositions and/or formulations areadministered to the subject at a dosage of active component(s) rangingfrom 0.1 µg/kg body weight to 100 g/kg body weight. In some embodiments,the disclosed compositions and/or formulations are administered to thesubject at a dosage of active component(s) ranging from 1 µg/kg to 10g/kg, from 10 µg/kg to 1 g/kg, from 10 µg/kg to 500 mg/kg, from 10 µg/kgto 100 mg/kg, from 10 µg/kg to 10 mg/kg, from 10 µg/kg to 1 mg/kg, from10 µg/kg to 500 µg/kg, or from 10 µg/kg to 100 µg/kg body weight.Dosages above or below the range cited above may be administered to theindividual subject if desired.

The subject can be any mammalian subject, for example a human, dog, cow,horse, mouse, rabbit, etc. In some embodiments, the subject comprises asmall molecule which can be sensed and responded to by the particle.

In some embodiments, the disease is glycemic imbalance. In someembodiments, the disease is hyperglycemia. In some embodiments, thedisease is diabetes.

In some embodiments, the method treats hyperglycemia by reducing bloodglucose levels. For example, the method can treat hyperglycemia byreleasing the therapeutic agent, wherein the therapeutic agent reducesblood glucose levels. In some embodiments, the method increases insulinlevels.

In some embodiments, the method avoids hypoglycemia by avoiding reducingblood glucose levels to hypoglycemic levels. For example, the method canavoid hypoglycemia by avoiding further releasing the therapeutic agentonce the desired glucose levels are achieved.

Also disclosed herein are methods to release insulin to an environmentcomprising increased glucose levels, the method comprising exposing tothe environment a particle comprising an inner liposomal vesicle (ILV)encapsulating insulin, an outer liposomal vesicle (OLV) encapsulatingthe ILV, a membrane fusion-promoting agent, and a pH-altering agent.Upon exposure of the particle to the environment comprising increasedglucose levels, the particle can release insulin encapsulated within theILV. Release can occur by mechanisms described herein, for instance byOLV-ILV fusion and subsequent “exocytosis” of encapsulated insulin.

The particle can be any particle disclosed herein.

The environment can be an artificial environment such as a laboratoryassay. Alternatively, the environment can comprise cells and/or tissues(e.g., in situ experiments or assays). In some embodiments, theenvironment can comprise a biological fluid, such blood or lymph. Insome embodiments, the environment can be within a subject, for instancea human.

In some embodiments, increased glucose levels are in comparison tophysiologically normal glucose levels.

EXAMPLES

The following examples are set forth below to illustrate thecompositions, methods, and results according to the disclosed subjectmatter. These examples are not intended to be inclusive of all aspectsof the subject matter disclosed herein, but rather to illustraterepresentative methods and results. These examples are not intended toexclude equivalents and variations of the present invention which areapparent to one skilled in the art.

Example 1 Materials and General Methods

Materials. All lipids including egg phosphatidylcholine (EPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),dipalmitoylphosphatidylcholine (DPPC),1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(ammonium salt), and1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-lissamine-rhodamine Bwere purchased from Avanti Polar Lipids, Inc. All peptides weresynthesized by Biochem Co., Ltd. (Shanghai, China). All DNAs were boughtfrom DNA Technologies, Inc. (Coralville, IA, USA). Cholesterol, glucoseoxidase, catalase, gramicidin A, methoxypolyethylene glycol maleimide(mPEG-Mal, M.W. = 5000), phenylmethylsulfonyl fluoride (PMSF), n-dodecylβ-D-maltoside (DDM) and 8-hydroxypyrene-1, 3, 6-trisulphonic acidtrisodium salt (HPTS) were purchased from Sigma-Aldrich. Humanrecombinant insulin was purchased from Life Technology. Anti-GlucoseTransporter 2 antibody was purchased from AβCam (cat. # ab54460).

Instrumentation. Circular dichroism (CD) spectra were recorded using aJasco J-815 spectropolarimeter (Jasco Inc., Easton, MD) while the samplecell was flushed with nitrogen. Transmission electron microscopy (TEM)images were acquired with JEOL 2000FX scanning transmission electronmicroscope (JEOL USA, Inc.) at 200 kV. Fluorescence measurements werecarried out by using a FLS980 fluorescence spectrophotometer (EdinburghInstruments). ELISA assays, insulin detection and cell viabilityanalysis were conducted on Infinite 200 PRO multimode plate reader(Tecan Group Ltd., Switzerland). Confocal microscopy images wereobtained with Zeiss LSM 710 confocal microscope (Carl Zeiss, Germany),and the samples were visualized using the same acquisition settings andanalyzed using Zen 2011 software (Carl Zeiss). Confocal microscopy moviewas recorded with Zeiss LSM 880 Airyscan. Cryogenic TEM images wereacquired with the help of ICBR Electron Microscopy at the University ofFlorida. The blood glucose levels in mice were monitored using theClarity GL2Plus glucose meter (Clarity Diagnostics, Boca Raton,Florida). Cryo-SEM images were obtained with JEOL 7600F equipped withGatan Alto (JEOL USA, Inc.). After cryo-facture of the larger liposomes,the sample was then sublimed at -95° C. for 5 mins under 10⁻⁶ mbarvacuum. This step was used to reveal the internal fine structure bysubliming the ice crystals. The darker background outside the largerliposomes was ice. From the differences in brightness, the ice and thesmall liposome nanoparticles can be distinguished.

Statistical analysis. Biological replicates were used in allexperiments. Student’s t-test or ANOVA were utilized to determinestatistical significance between different groups. A P value <0.05 wasconsidered to be statistically significant. All statistical analyseswere performed using Origin 8.5. No statistical methods were used topre-determine the sample size of the experiments.

Design and Synthesis of Artificial β Cells (AβCs)

Synthesis of PEG shield. 3'-thiolated cytosine (C)-rich DNA (CNDA) andmPEG-Mal solution at a DNA/mPEG-Mal molar ratio of 1/5 were mixed inHEPES buffer (1 mM, pH 7.5) and incubated for 4 h. The conjugation ofPEG with CDNA (PEG-CDNA) was verified by agarose gel (1%)electrophoresis (FIG. 6 panel a), where PEG-CDNA showed a much slowermobility than CDNA. After that, the cholesterol-ended guanine (G)-richDNA (GDNA-CH) was mixed with PEG-CDNA to form duplexed DNA bridged PEGshield (PEG-CDNA/GDNA-CH), which had the capability to insert intoliposome membranes. The fluorescently labeled DNA and non-pH-responsiveDNA bridged PEG shield was prepared by the same process. The DNA bridgedPEG shields were lyophilized before preparation of liposomes. Thesequences of all DNAs are shown in Table 1. The conformational switch ofthe PEG-CDNA/GDNA-CH and non-pH-responsive DNA bridged PEG shield atdifferent pH was studied by the CD spectroscopy (FIG. 7 ). From the CDspectra, no conformational variation was observed for the control DNA.However, for PEG-CDNA/GDNA-CH the characteristic peak of DNA tetraplexaround 290 nm increased as the pH decreased. The CD spectra ofPEG-CDNA/GDNA-CH showed that the PEG-CDNA/GDNA-CH mainly existed asduplex at and above pH 6.5, whereas the PEG-CDNA/GDNA-CH disassociatedto form tetraplex at and below pH 5.5.

Preparation of insulin-loaded fusogenic small liposomal vesicles (ISV)labeled with peptide-K and PEG shield. Lipid stock solution was preparedin chloroform with the composition of EPC/DOPE/CH (2.5:2.5:1, w:w:w).Peptide-K (50 µM) and PEG shield (50 µM) stock solutions were preparedin chloroform/methanol (1:1, v:v) and methanol, respectively. Then asolution of lipids, peptide-K and PEG shield was prepared with finalmolar ratio of 95:1:4 and added to round-bottom flask. Afterwards, thesolvent was evaporated by the rotary vacuum evaporator to get lipid filmand the lipid film was dried under vacuum overnight. Next, insulin wasdissolved in 4 mL HEPES buffer (5 mM, 150 mM NaCl, pH 7.4) and added torehydrate the lipid film with gently vortex. A total 50 mg of insulinwas used for each 100 mg of lipid. The resulting suspension wassonicated by a probe sonicator for 5 minutes with a power of 100 W andtime interval of 1 s/1 s to get a milky suspension. Afterwards, thesuspension was extruded 10 times through polycarbonate membranes withpore size of 100 nm using an Avanti mini-extruder (Avanti Polar Lipids,Alabaster, AL). The free insulin was removed by centrifugation at 20,000rpm for 60 min at 4° C. and the liposomes were washed twice. To thesedimented liposomes, 4 mL HEPES buffer was added to get the finalinsulin-loaded ISV. The loading efficiency of insulin was calculated asthe ratio of the concentrations of the liposome-associated insulin tothe initially added insulin and it was approximately 36.4% by aCoomassie Plus protein assay. ISV without insulin or peptide-K wereprepared by the same manner and used in control experiments.

Preparation of interdigitated DPPC bilayer sheets (Kisak, E., et al.,Langmuir 18, 284-288 (2002); Boni, L.T. et al., BBA-Biomembranes 1146,247-257 (1993); Ahl, P. et al., BBA-Biomembranes 1195, 237-244 (1994)).DPPC dissolved in chloroform and peptide-E dissolved inchloroform/methanol (1:1, v:v) at a molar ratio of 98:2 were added to around-bottom flask. After the solution was evaporated to form thin lipidfilms, HEPES buffer was added and the film was hydrated at 60° C. Thefinal lipid concentration was 50 mg/mL. Afterwards, the suspension wassonicated at 60° C. for 5 min with a power of 100 W and time interval of1 s/1 s to get a milky suspension. Then, the solution was extrudedsequentially through syringe filters with pore size of 200 and 100 nmfor 10 times respectively at 60° C. to get liposomes with size less than100 nm (FIG. 10 panels a, b). Afterwards, interdigitated sheets wereformed by adding ethanol dropwise to 0.5 mL of the DPPC liposomes; thefinal concentration of ethanol was typically 3M. The solution wasallowed to sit for 2 h to ensure complete interdigitation. After that,the resulting sheets were washed thrice by adding 3 mL HEPES buffer toremove excess ethanol, followed by centrifugation at 5000 rpm andremoval of supernatant. The obtained sheets were characterized byCryo-SEM (FIG. 10 panel c) and transmission electron microscopy (FIG. 10panel d).

Building AβCs: To encapsulate ISVs inside the outer large liposomalvesicles (OLVs), 0.5 mL of the above prepared ISVs, 6 mg glucose oxidaseand 1.5 mg catalase were added to one batch of the above prepared DPPCsheets. Then, the mixture was heated at 45° C. (above the maintransition temperature of DPPC) for 20 min under gentle stirring todrive the sheets to close around ISV and glucose oxidase/catalase toform the vesicles-in-vesicle superstructures (ISVs@OLV) (Kisak, E., etal., Langmuir 18, 284-288 (2002); Boni, L.T. et al., BBA-Biomembranes1146, 247-257 (1993); Ahl, P. et al., BBA-Biomembranes 1195, 237-244(1994)). The free ISV and glucose oxidase/catalase were removed bycentrifugation at ~2000 rpm and washed twice. All the supernatant wascollected and ultracentrifuged to measure the loading efficiency of ISVand glucose oxidase/catalase. The amount of the ISV encapsulated insideOLV was determined indirectly by measuring the amount of insulin in thefree ISV. About 52.8% of ISV were encapsulated. The unencapsulatedglucose oxidase/catalase was detected by Bradford staining to measurethe protein amounts in the supernatant after ultracentrifugation. About45.2% of glucose oxidase/catalase were encapsulated. After that, glucosetransporter 2 (expression and purification methods shown below) wasreconstituted onto the OLV according to a previously reported method(Kasahara, M. et al., Proc. Natl Acad. Sci. USA 73, 396-400 (1976)).Approximately 1 mg glucose transporter 2 in was mixed with 1/5 of thecentrifuged ISVs@OLV in 0.5 mL HEPES buffer. After mixing homogeneously,the suspension in a tube was quickly frozen in liquid nitrogen for 5 minand subsequently sonicated for 20 or 30 s in a bath sonicator. Thesuspension was centrifuged at ~2000 rpm and washed twice to remove freeglucose transporter 2. The reconstitution efficiency of glucosetransporter 2 was determined as 82% by the glucose transporter 2 ELISAKits. Finally, the proton channel Gramicidin A (GA) at differentGA-to-DPPC ratios in DMSO solution was added to the suspension ofglucose transporter 2-recontituted ISVs@OLV used for the followingexperiments.

Glucose transporter 2 expression and purification. The cDNA encodingmouse glucose transporter 2 (Origene, Genbank accession # NM_031197) wasamplified and cloned into the BamH I and Hind III sites of pET-28a(Novagen, see Supplementary Note 1 for full plasmid sequence). Theconstructed plasmid was transfected into E. coli Rosetta (DE3) pLysScells for glucose transporter 2 expression. The E. coli cells werecultured in lysogeny broth supplemented with kanamycin andchloromycetin, protein expression was induced with 0.5 mM isopropylβ-D-1-thiogalactopy-ranoside. The cells were collected by centrifugationand re-suspended in Buffer A (20 mM Tris-HCl pH 8.0, 0.15 M NaCl, 10 mMimidazole, 1 mM PMSF, 5% glycerol, 0.4 mg/mL DNase I, 2% DDM and 0.5mg/mL lysozyme). The suspension was incubated at 25° C. for 0.5 h andkept on ice for another 0.5 h. After brief sonication, cell debris wasremoved by centrifugation (20000 ×g, 10 min). The clear supernatant wasadded to a column filled with Ni-NTA resin (Qiagen). After washing thecolumn with Buffer B (20 mM Tris-HCl, pH 8.0, 25 mM imidazole, 5%glycerol, 0.15 M NaCl and 0.05% DDM), glucose transporter 2 was elutedwith Buffer C (20 mM Tris-HCl, pH 8.0, 0.15 M NaCl and 500 mM imidazole,5% glycerol and 0.05% DDM). Purified glucose transporter 2 wasquantified by the Bradford staining (Bio-Rad) and the purity wasanalyzed by SDS-PAGE (FIG. 13 ).

Biochemical Processes inside AβCs

Glucose sensing and metabolism by AβCs. The fluorescence intensities ofHPTS (514 nm emission, 406 (I₄₀₆) and 460 (I₄₆₀) nm excitation) werestrongly dependent on the degree of ionization of the 8-hydroxyl group(pK_(a) = 7.2) and hence on the pH of the medium. The intensities ofI₄₀₆ and I₄₆₀ increased linearly with increasing HPTS concentrations upto 2 µM (Tunuguntla, R.H., et al., Nat. Nano. 11, 639-644 (2016); Kano,K. et al., BBA-Biomembranes 509, 289-299 (1978)). The titration curve ofHPTS (0.5 µM) at different pH (HEPES buffer, 5 mM, 100 mM NaCl) wasmeasured as shown in FIG. 14 panel a. For glucose sensing, HPTS wasloaded with ISV/GOx/CAT inside the OLV simultaneously during theencapsulation process and was used as the pH probe to measure the pHvariation inside AβC as induced by glucose uptake, glucose oxidation andproton efflux. To keep a physiologically-relevant and constant externalglucose concentration, 100 µL HEPES buffer containing HPTS loaded AβCsand glucose at different concentrations were added into the cup of aSlide-A-Lyzer™ MINI Dialysis Device (2K) and 1.5 mL isotonic solutioncontaining the same concentration of glucose was added to the tube.After that, the Mini Dialysis Device was gently shaken at 37° C. Atpredetermined time intervals, 30 µL of the solution in the cup was takenout and the fluorescence intensities at 510 nm excited by 400 and 450 nmwere detected. The pH values inside AβC were calculated using thetitration curve shown in FIG. 14 panel b. After measurement, thesolution was returned back to the cup of Min Dialysis Device. For theglucose concentration switch experiments, AβC was first incubated athigh glucose concentrations to reach equilibrium, and then the test ofAβC’s ability towards sensing changed glucose levels started. After eachswitch, the inner pH was measured until reaching equilibrium and thenthe AβCs were centrifuged and then re-suspend in another glucosesolution. The cycles were repeated several times.

‘Signal transduction’ inside AβC. The PEG shield attachment to anddissociation from the ISV surface triggered by pH variation induced byglucose metabolism was investigated. As for AβC, there were proteins orpeptides that also had CD signals, fluorescence resonance energytransfer (FRET) method was alternatively applied to monitor this processby labeling fluorescent dye (tetramethylrhodamine, DNA-donor) onto CDNAand quencher (IAbRQ, DNA-acceptor) onto GDNA (Table 1). Similar to theglucose sensing experiments, the AβC solution was taken out atpredetermined time-intervals or cycle runs and the fluorescencevariation of DNA-donor at different glucose concentrations wasdetermined. To study the PEG shedding-facilitated peptide assembly ofpeptide-E on ISV with peptide-K on the inner surface of OLV, the FRETmethod was also employed by using lysine-nitrobenzofuran(peptide-donor)-modified peptide-K andlysine-5-carboxytetramethylrhodamine (peptide acceptor)-labeledpeptide-E. The interaction of the peptide was determined by thequenching efficiency of the peptide-donor by the peptide acceptor atdifferent glucose concentrations.

Membrane Fusion

The fusion of ISV with OLV in response to different glucoseconcentrations was studied by a dequenching method based on FRET.Fluorescence experiments were carried out using ISV labelled with theFRET pair1,2-dioleoyl-snglycero-3-phospoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(ammonium salt) (lipid-donor, 0.5 mol%), and1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-lissamine-rhodamine B(lipid acceptor, 0.5 mol%) co-labeled ISV. After incubating AβC indifferent glucose solutions, the recovery of lipid-donor fluorescencewas detected at different time intervals. The change in lipid-donorfluorescent intensity was plotted as:

F(%) = 100 × (F_(t)-F₀)/(F_(max)-F₀)

where F₀ was the lipid-donor fluorescent intensity at t=0 beforeaddition to glucose solution, F_(t) was the lipid-donor fluorescentintensity measured at time t, and F_(max) was the lipid-donorfluorescent intensity after disruption of the AβC in 1% (w/v) TritonX-100. Also, the fusion process was monitored by confocal lasermicroscopy imaging (Zeiss LSM 710). Images were taken at different timeafter incubation in glucose solution.

For experiments demonstrating that no hemisfusion stage existed, theouter fluorescence of lipid-donor/lipid-acceptor co-labeled ISV wasbleached by incubation with sodium dithionate (FIG. 23 ). The excesssodium dithionate was removed by centrifugation at 20000 rpm. Then, thefusion of the inner membrane of ISV with the outer membrane of OLV wasdetected by an identical dequenching assay.

In Vitro Dynamic Insulin ‘Secretion’ from AβCs

100 µL AβC mixed with different concentrated glucose solutions wereadded into the cup of a Slide-A-Lyzer™ MINI Dialysis Device (2K) and 1.5mL isotonic solution containing the same concentration of glucose wasadded to the tube. After that the Mini Dialysis Device was gently shakenat 37° C. At indicated time points, 15 µL of the solution in the cup wastaken out and centrifuged at 6000 rpm for 1 min. Then certain volume ofthe supernatant was taken out and the insulin was measured by theCoomassie Plus protein assay. The concentration was calculated with aninsulin standard curve (FIG. 25 ). For concentrated samples, dilution tothe range of the standard curve was needed. The remaining mixture wasresuspended in paramount of fresh glucose solution and re-added to thecup. For pulsatile release studies, the AβC was first incubated within400 ml/dL solution for 60 min and then was spun down by centrifugationand washed once with HEPES buffer. After that, the AβC was re-suspendedin 100 mg/dL glucose solution for another 60 min. The cycles wererepeated numerous times. Similarly, insulin concentration was determinedusing the Coomassie Plus protein assay.

In Vivo Diabetes Treatment

Animal. Animal experiments were performed according to the animalprotocol approved by the Institutional Animal Care and Use Committee atthe University of North Carolina at Chapel Hill and North Carolina StateUniversity. Streptozotocin (STZ)-induced C57BL/6 type 1 diabetic micewere purchased from the Jackson Lab. Experimental group sizes wereapproved by the regulatory authorities for animal welfare after beingdefined to balance statistical power, feasibility and ethical aspects.For all the animal studies, mice were randomly allocated to each group.The researchers were not blinded to group allocation during the animalstudies, as demanded by the experimental designs.

Diabetes treatment. The in vivo efficacy of AβC was investigated on theSTZ-induced adult diabetic mice (male, age 8 week). The blood glucoselevel of mice was monitored two days before ‘transplantation’ of the AβCusing the Clarity GL2Plus glucose meter (Clarity Diagnostics, BocaRaton, Florida) by collecting ~3 µL blood from the tail vein, and allmice were fasted overnight before administration. To mimic β-celltransplantation, AβC was suspended into 40% PF127 solution andsubcutaneously injected into the dorsum of diabetic mice to form athermogel (FIG. 30 ). A total of 300 µL of AβC/PF127 in PBS buffer orother control mixture was subcutaneously injected into the dorsum ofmice with an insulin dose of 50 mg/kg after anesthesia with isoflurane.Five mice with stable hyperglycemic state were chosen for each group to‘transplant’ AβC, AβC_((no) _(insulin)), control AβCs that eitherlacking glucose sensing machinery (GSM) or membrane fusion peptides(AβC_((no) _(GSM)) and AβC_((no) _(PE/PK))). The blood glucose levelswere monitored over time (every 15 min or 1 h for the first 12 h afteradministration and once per day in the following days) until returningto stable hyperglycemia. To confirm the bioactivity of the releasedinsulin, insulin solution (20 µg of native insulin or insulin releasedfrom AβC at 400 mg/dL glucose for 6 h.) was subcutaneously injected intothe dorsum of diabetes mice. To measure the plasma insulin level invivo, 25 µL of blood sample was collected from the tail vein of mice atindicated time points. The plasma was isolated and stored at -20° C.until assay. The plasma insulin concentration was measured using a HumanInsulin ELISA kit according to the manufacturer’s protocol (Calbiotech,USA). A series of glucose tolerance tests were conducted at 24, 36 and48 h post transplantation to confirm the effective release of insulinfrom the AβCs in response to the glucose challenge. Briefly, glucosesolution in PBS was intraperitoneally injected into all mice at a doseof 1 g kg⁻¹. The blood glucose level was closely monitored for 120 minafter injection. The results of glucose tolerance tests on healthy micewere used as control.

Cell culture. HeLa cells were obtained from Tissue Culture Facility ofUNC Lineberger Comprehensive Cancer Center. The cells were cultured inDulbecco’s Modified Eagle Medium (Gibco, Invitrogen) with 10% (v:v)fetal bovine serum (Invitrogen), 100 U ml⁻ ¹ penicillin (Invitrogen) and100 mg ml⁻¹ streptomycin (Invitrogen) in an incubator (ThermoScientific) at 37° C. under an atmosphere of 5% CO₂ and 90% relativehumidity. Cells were tested every three months for potential mycoplasma.Re-authentication of cells was not performed after receipt.

Biocompatibility evaluation. The cytotoxicity of AβC_((no) _(insulin))was examined on HeLa cells by a 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay.Briefly, HeLa cells were seeded at a density of 5000 cells well⁻¹ (200µL total volume well⁻¹) in 96-well plate. After 24 h, AβC_((no)_(insulin)) at indicated concentrations was added and cells were furtherincubated for 24 h. Thiazolyl blue solution (5 mg mL⁻¹) was added intowells (final concentration: 0.5 mg mL⁻¹) and incubated with cells for 4h. After removing the culture media, the purple formazan crystal wasdissolved in 200 µL of DMSO. The absorbance at 570 nm, which is directlyproportional to the viable cell number, was measured on the Infinite 200PRO multimode plate reader (Tecan Group Ltd., Switzerland).

To evaluate the in vivo biocompatibility of AβC, four weeks afterinjection of AβC/PF127, the diabetes mice were euthanized via CO₂asphyxiation, and the injected materials and surrounding tissues wereexcised. The tissues were then fixed in 10% formalin, embedded inparaffin, cut into 5 µm sections, and stained using hematoxylin andeosin for histological analysis. During the same time, the body weightof the diabetes mice in each group was recorded. Moreover, for immuneanalysis, representative inflammatory factors, such as TNF-a, IL-1β andIL-6, in the serum of each group were measured by ELISA according to themanufacturer’s instructions.

TABLE 1 DNA sequences used for synthesis of PEG shield. pH-responsiveDNA CDNA 5’-CCC TTA CCC TTA CCC TTA CCC TTT TTT-SH-3’ SEQ ID NO:3TAMRA-labeled CDNA 5’-CCC TAA CCC TAA CCC TAA CCC T/i5-TAMK/T TTTT-SH-3’ SEQ ID NO:4 *GDNA 5’-GGG TTA GGG TTA GGG TTA GGG TTT TTT-CH-3’SEQ ID NO:5 IAbRQ-labeled GDNA 5’-IAbRQ-GGG TTA GGG TTA GGG TTA GGG TTTTTT-CH-3’ SEQ ID NO:6 Non-pH-responsive DNA DNA1 5’-CTC TCA CAC TCA CTCTCA CGC TTT TTT-SH-3’ SEQ ID NO:7 TAMRA-labeled DNA1 5’-CTC TCA CAC TCACTC TCA CGC T/i5-TAMK/TT TTT-SH-3’ SEQ ID NO:8 DNA2 5’-GCG TGA GAG TGAGTG TGA GAG TTT TTT-CH-3’ SEQ ID NO:9 IAbRQ-labeled DNA2 5’-IAbRQ-GCGTGA GAG TGA GTG TGA GAG TTT TTT-CH-3’ SEQ ID NO:10

Results

Construction of the AβCs. To implement the AβC design, insulin-loadedISVs were first prepared via the classic lipid film hydration method(Mo, R., et al., Angew. Chem. Int. Ed. 53, 5815-5820 (2014)), where 4mol% PEG-CDNA/GDNA-CH (FIG. 6 ) and 1 mol% peptide-K were inserted intothe membrane by in situ modification. FIG. 1 panel b presents atransmission electron microscopy image of the ISVs, which have anaverage diameter of 50 nm. Due to the conformational switch betweendouble-stranded duplex DNA and intramolecular tetraplex structures atphysiological pH and acidic pH (pH<6.0, FIG. 7 ) (Zhao, C., et al.,Chem. Eur. J. 17, 7013-7019 (2011); Li, X., et al., Proc. Natl Acad.Sci. USA 103, 19658-19663 (2006)), the PEG shield was reversiblyattached to and disassociated from ISV, as shown by the fluorescentresonance energy transfer (FRET) method (FIG. 8 ). The peptide-K and thepeptide-E were each designed with three features: a transmembranedomain, a spacer, and a recognition motif (FIG. 9 panel a), mimickingthe membrane fusion SNARE polypeptides (Marsden, H. et al., Chem. Soc.Rev. 40, 1572-1585 (2011); Lygina, A. et al., Angew. Chem. Int. Ed. 50,8597-8601 (2011); Robson Marsden, H., et al., Chem. Int. Ed. 48,2330-2333 (2009); Meyenberg, K., et al., Chem. Commun. 47, 9405-9407(2011); Tomatsu, I. et al., J. Mater. Chem. 21, 18927-18933 (2011);Kong, L., et al., Angew. Chem. Int. Ed. 55, 1396-1400 (2016); Gong, Y.,et al., J. Am. Chem. Soc. 130, 6196-6205 (2008); Chan, Y. et al., Proc.Natl Acad. Sci. USA 106, 979-984 (2009); Steinmetz, M. et al., Proc.Natl Acad. Sci. USA 104, 7062-7067 (2007)). As such, heterodimericcoiled coils driven by the electrostatic and hydrophobic interactions ofthe heptad repeats in the recognition part were formed upon mixingpeptide-E and peptide-K (FIG. 9 panel b). Insertion of the pH-responsivePEG shield on ISVs, an interaction between peptide-E and peptide-Koccurred only in the unshielded state (Tomatsu, I. et al., J. Mater.Chem. 21, 18927-18933 (2011); Kong, L., et al., Angew. Chem. Int. Ed.55, 1396-1400 (2016)). ISVs and glucose oxidase/catalase wereencapsulated into OLV decorated with 2 mol% peptide-K by heatinginterdigitated dipalmitoylphosphatidylcholine sheets above thegel-liquid phase transition temperature (FIG. 10 ) (Kisak, E., et al.,Langmuir 18, 284-288 (2002)). Annealing the interdigitated phospholipidsheets led to the formation of closed compartments with large size andhigh internal volume (Kisak, E., et al., Langmuir 18, 284-288 (2002)),simultaneously entrapping enzymes and ISVs at high efficiency. Catalasewas added to decompose the undesired hydrogen peroxide and regenerateoxygen to facilitate glucose oxidation (FIG. 11 ). The resultingvesicles-in-vesicle superstructures with an overall size of ~ 1-5 µm wasshown via cryogenic scanning electron microscopy (Cryo-SEM) image (FIG.1 panels c, d), cryogenic transmission electron microscopy image (FIG.12 ), and a confocal microscopy movie. The localization glucoseoxidase/catalase inside the cavities of OLV was demonstrated by confocallaser microscopy imaging (CLSM; FIG. 1 panel e). Next, glucosetransporter 2, which was expressed and purified from E. coli (FIG. 13panel a), was reconstituted into the OLV membrane by a freeze-thawsonication method. The presence of glucose transporter 2 on the OLVmembrane was verified by Western blotting (FIG. 1 panel f, FIG. 13 panelb) and CLSM imaging (FIG. 1 panel g). Finally, gramicidin A, amembrane-spanning channel capable of conducting protons at very highflux rates (Tunuguntla, R.H., et al., Nat. Nano. 11, 639-644 (2016)),was inserted into the membrane of OLV, as shown in the CLSM image byincorporating lysine-5-carboxyfluorescein-ended gramicidin (FIG. 1 panelh).

Biochemical processes inside AβCs. To determine the glucose sensingability of the AβCs, the glucose transport machinery was coupled to aninterior enzymatic oxidation scheme and a pH-sensitive dye (FIG. 14 ).The biochemical process was monitored by fluorescence measurements(FIGS. 2A-2B, FIG. 15 panels a-b). At a fixed lipid/glucosetransporter/glucose oxidase ratio, the inner pH level was dependent onthe external glucose concentration as well as the amount of addedgramicidin. At the same gramicidin level, the pH decrease wassignificantly faster at 400 mg/dL glucose (a typical hyperglycemialevel) than that at 100 mg/dL (a normoglycemia level). Moreover, similarpH variations but with relatively slower kinetics were observed atmodest hyperglycemic levels (300 and 200 mg/dL). Glucose uptake byglucose transporter was confirmed in control systems with no glucosetransporter present as well as in the presence of theinhibitor-cytochalasin B (FIG. 16 ). Given that glucose transporter 2has a K_(m) of about 15-20 mM (Efrat, S., Nat. Genet. 17, 249-250(1997)) and glucose oxidase has a K_(m) of about 33-100 mM, thedifference in pH decline was attributed to the different glucosetransport rates associated with high and low glucose concentrations.When the glucose concentration was held constant, inner pH was inverselycorrelated to the content of gramicidin; the more gramicidin wasinserted, the faster protons were pumped out of the AβCs. In controlsystems where no gramicidin was inserted, the final pH at 100 mg/mL wassimilar to that at hyperglycemic levels, although the former decreasedslower, indicating the necessity of gramicidin for tuning proton effluxto distinguish high and low glucose concentrations. The maximumpH-change therefore relied on the overall kinetics of glucose uptake,glucose oxidation and proton efflux. For the following studies, AβCswith gramicidin-to-lipid ratio of 1:2000 having capacity to maintain pH< 5.6 at hyperglycemic level and pH > 6.5 at normoglycemia level wereselected. In this system, pH inside the AβC was reversed in response toadjustment of glucose concentration (FIG. 2C, FIG. 15 panels c-d).Notably, fast response in switching pH was due to the fact that thediffusion of intermediates was minimized since all the biochemicalprocesses were confined in a micron-scale space (Matsumoto, R. et al.,Phys. Chem. Chem. Phys. 12, 13904-13906 (2010)). Such unique propertiesfacilitate the artificial system’s ability to act like natural β cellsin terms of precisely sensing graded glucose levels.

Next, ‘signal transduction’ inside AβCs including the steric PEGdeshielding and the subsequent peptide-E/peptide-K assembly wasinvestigated. The disassociation of the PEG shield induced by glucosemetabolism was confirmed via a FRET assay (FIG. 2D). The emission at 590nm of the tetramethylrhodamine (DNA-donor) on PEG-CDNA, which wasinitially quenched by IAbRQ (DNA-acceptor) on GDNA-CH, graduallyincreased in 200-400 mg/dL glucose solutions, whereas almost no changewas observed for that in 100 mg/dL glucose solution (FIG. 2E, FIG. 17panel a). Due to the slow kinetics for pH variation at modesthyperglycemic levels, the corresponding changes in PEG deshielding tooka relatively longer time to initiate in 200 and 300 glucose solutions. Acontrol study of a pair of randomly sequenced complementary DNA strandgenerated no fluorescence recovery at either high or low concentrationsover time (FIG. 18 ). Furthermore, reversible association anddisassociation of the PEG-CDNA was observed when the glucoseconcentrations were cyclically varied between high glucose levels and100 mg/dL (FIG. 2F, FIG. 17 panels b-c), indicating that theconformational conversion at high glucose levels induced the duplexdehybridization and hence the detachment of the PEG shield.

Next the pairing of peptide-K on ISV with the peptide-E on OLV wasstudied. Because signals of other peptides/proteins prohibited the useof circular dichroism techniques, FRET experiments were used (FIG. 2G).As shown in FIG. 2H and FIG. 19 panel a, the fluorescence ofnitrobenzofuran on peptide-K (peptide-donor) was readily quenched by thetetramethylrhodamine (peptide-acceptor) on peptide-E at high glucoseconcentrations, and a stepwise decrease in peptide-donor fluorescencewas observed by switching the glucose concentration between high glucoselevels and 100 mg/dL (FIG. 2I, FIG. 19 panels b-c). The lag at thebeginning of each switch was due to the inner pH variation and the timerequired for the PEG shield attachment/detachment to reach equilibrium;this lag was longer in the cases of modest hyperglycemic solutions thanafter exposure to the high glucose concentration. Critically, no peptideinteractions were detected at low glucose levels or in control groupswhere non-pH responsive DNA was used (FIG. 20 ), indicating the directdependence of peptide assembly on the dissociation of the PEG shieldfrom ISVs, where protons generated by glucose (signal) metabolism act asthe signal mediator and the sequence-specific DNA strands act as thesignal effector facilitating the precise signal transduction inside AβCto induce PEG deshielding and subsequently activate peptide assembly.

Membrane fusion of ISV with OLV. With a clear ‘signaling pathway’, thenext step was to substantiate the membrane fusion of the ISV with OLVdriven by the interaction of peptide-E and peptide-K. By simultaneouslyincorporating lipids labelled with nitrobenzofuran (lipid-donor) andlissamine rhodamine B (lipid-acceptor) into the ISV, the efficiency oflipid mixing at different glucose concentrations was studied by astandard dequenching assay (FIG. 3A). At high glucose concentrations,continuous increase in lipid-donor emission around 536 nm was observed,indicative of dilution of the lipid-donor and lipid-acceptor dyesinduced by membrane fusion (FIG. 3B, FIG. 21 panel a). Also, as can beseen in the CLSM image (FIG. 3C), the green fluorescence of lipid-donorand red fluorescence of lipid-acceptor gradually appeared and increasedon the surface of AβC in response to hyperglycemic conditions, and thefluorescence inside the AβCs gradually decreased, showing the merger ofthe ISV with the OLV. No obvious lipid-donor fluorescence variation wasobserved under normal glycemic level (FIG. 3D). In further controlexperiments, no recovery in lipid-donor fluorescence was detected wheneither peptide-E or peptide-K was omitted (FIG. 22 ). Aspeptide-E/peptide-K has been reported to be the minimal machinery thatcan mimic SNARE polypeptides for controlling membrane fusion (RobsonMarsden, H., et al., Chem. Int. Ed. 48, 2330-2333 (2009); Meyenberg, K.,et al., Chem. Commun. 47, 9405-9407 (2011)), these results thereforeshowed that glucose signal transduction-tuned assembly of the peptidesbrought the ISV and OLV closely together and transmitted the forces forpromoting their fusion.

In natural beta cells, vesicles can be trafficked to the periphery ofthe cell membrane with the assistance of the cytoskeleton (Wang, Z. etal., J. Cell Sci. 122, 893-903 (2009)), leading to a sustainable fusionprocess. Lacking machinery mimicking the cytoskeleton, ISV movedrandomly by Brownian motion within AβC, as shown in CLSM images (FIGS.3C-3D). Both the peptide assembly and the effective contact between therandomly diffused ISVs and the OLV contributed to the observed fusionprocesses within AβCs (Schuette, C. et al., Proc. Natl Acad. Sci. USA101, 2858-2863 (2004)), making the fusion process sustainable forseveral hours. In addition, cyclic switches in the solution glucoseconcentrations between hyperglycemic and normoglycemic levels resultedin stepwise increases in nitrobenzofuran fluorescence (FIG. 3E, FIG. 21panels b-c). Thus, based on the glucose sensing ability of AβCs and thereversible attachment of the PEG shield on ISV, this newglucose-responsive mechanism promotes fusion under high glycemicconditions and reshields the ISVs to minimize fusion at normoglycemiclevels for numerous cycles.

To exclude the possibility that the fusion process was terminated at thestage of hemifusion (Lygina, A. et al., Angew. Chem. Int. Ed. 50,8597-8601 (2011); Robson Marsden, H., et al., Chem. Int. Ed. 48,2330-2333 (2009); Meyenberg, K., et al., Chem. Commun. 47, 9405-9407(2011)), it was investigated whether the inner lipid layers of ISVeffectively mixed with the outer lipid layers of OLV by bleaching thelipid-donor fluorophores on the outside of the ISV with sodiumdithionite (FIG. 23 panel a). Still, an increase in lipid-donorfluorescence was detected at 400 mg/dL glucose (FIG. 23 panel b). Sincethe FRET effect needed the lipid-donor fluorophores inside the ISV,therefore, complete merger of both the inner and outer lipid leaflets ofthe membranes of ISV and OLV occurred during fusion. Such full fusion ofISV with OVL is important for the release of ISV contents outside theOLV in a way that mimics cellular exocytosis.

In vitro dynamic insulin ‘secretion’ from AβCs. In accumulated insulinrelease studies, a remarkably rapid insulin release was detected athyperglycemic levels while minimal release was observed at a normalglucose level or in glucose-free buffer solution over 15 h (FIG. 4A,FIG. 24 , and FIG. 25 panel a). Notably, the fast responsiveness of AβCsto 400 mg/dL glucose compared to more conventional glucoseoxidase-mediated pH decrease-dependent systems was due to thewell-organized spatial confinement of all relevant biochemicalprocesses, similar to compartmentalization in natural cells, and alsoreduced the interference from the buffered environment (Veiseh, O. etal., Nat. Rev. Drug. Discov. 14, 45-57 (2015); Mo, R., et al., Chem.Soc. Rev. 43, 3595-3629 (2014)). Meanwhile, the slower insulin releaseat modest glucose levels further contributes to dynamic response of AβCstowards different blood glucose levels. When insulin was conjugated withfluorescein isothiocyanate, CLSM imaging visualized that morehomogenously distributed green fluorescence was detected for solutionscontaining AβCs high glucose solutions while clustered green signalsremained at 100 mg/mL after incubation for 6 h (FIG. 4B, FIG. 25 panelb), substantiating the release of insulin under hyperglycemicconditions. In control groups where AβCs lacked either glucose sensingmachinery or membrane fusion peptides, very slow and non-distinguishableinsulin release kinetics were observed at all glucose levels (FIG. 26panels a, b). Furthermore, when cyclically alternating the glucoseconcentrations between 400 mg/dL and 100 mg/dL every 1 h for severalrepetitions, a pulsatile release profile was measured for AβCs, with amaximum of 8-fold difference in insulin release (FIG. 4C). The lowlevels of released insulin detected after switching the glucoseconcentration to normoglycemic level was due to the lag between glucosemetabolism, pH variation, and membrane fusion. However, no suchoscillations were found for the control AβCs (FIG. 26 panels c, d). Inaddition, no readily detectable insulin release was observed under mildacidic pH’s (FIG. 27 ), avoiding non-specific activation of AβCs indietetic mice suffering from diabetic ketoacidosis which typically leadsto plasma pH level below 7.3 (KitApChi, A. et al., Diabetes Care 32,1335-1343 (2009)). These results illustrated that the oscillations inglucose pathway and membrane fusion of AβCs effectively contributed tothe oscillatory feature of insulin release, closely mimicking thedynamic secretion features of natural β-cells.

In vivo type 1 diabetes treatment. The ability of AβC for regulatingblood glucose levels in vivo in a streptozotocin-induced type 1 diabeticmice model was examined. Prior to in vivo studies, the retention of thesecondary structure and the bioactivity of the insulin released fromAβCs were verified (FIG. 28 and FIG. 29 ). The AβCs were ‘transplanted’into the subcutaneous tissues by injecting a Pluronic F-127 (PF127)solution (40 wt%) containing homogenous distributed AβCs, as PF127 isbiodegradable and can quickly form a stable hydrogel at body temperature(FIG. 30 ) (Park, M.H., et al., Accounts Chem. Res. 45, 424-433 (2012)).The distribution and integrity of AβCs inside the hydrogels was shownvia Cryo-SEM imaging (FIG. 31 ). In control groups, AβCs lacking insulin(AβC_((no) _(insulin))), lacking the membrane fusion peptides (AβC_((no)_(PE/PK))) and lacking glucose sensing machinery (AβC_((no) _(GSM)))were also ‘transplanted’. The blood glucose levels in mice‘transplanted’ with AβC quickly declined from hyperglycemia tonormoglycemia within 1 h, and after that, the blood glucose levelremained normoglycemic for up to five days (FIGS. 4D and 4E). Incontrast, mice ‘transplanted’ with AβC_((no) _(insulin)) showed elevatedblood glucose levels, excluding the possibility of blood glucose leveldecrease induced by catalytic consumption. For groups treated withcontrol AβCs, elevated blood glucose levels were also observed due tothe lack of self-regulated glucose sensing or membrane fusion abilities.Correspondingly, plasma insulin levels in mice treated with AβCsremained detectable over the time course while little or no plasmainsulin was detected in the plasma of control groups (FIG. 4F).Intraperitoneal glucose tolerance tests were performed to further testthe in vivo glucose responsive ability of AβCs at 24, 36, and 48 h post‘transplantation’ (FIG. 4G). Following a spike in blood glucose leveleach time, mice which had received AβCs showed restoration ofpre-challenge blood glucose levels at a rate comparable to that inhealthy mice. Similar results were also observed on the fifth day after‘transplantation’ (FIG. 32 ). However, no such phenomenon was observedfor groups ‘transplanted’ with control artificial cells in the same test(FIG. 33 ). This in vivo responsive ability of the designed AβCs wasfurther proved by detecting the protein release in wild-type mice thatwere ‘transplanted’ with human serum albumin-loaded AβCs 1.5 h afteradministration of glucose (FIG. 34 ).

Importantly, AβCs_((no) _(insulin)) were not associated with readilydetectable cytotoxicity at any of the concentrations studied (FIG. 35 ).Finally, the transplanted formulation including PF127 was completelydegraded by four weeks post-administration, with no noticeableinflammatory region or fibrotic encapsulation (FIG. 36 ). At the sametime, no obvious differences in body weight change were observed betweentreated groups and the blank group injected with PBS (FIG. 37 ). Also,the levels of inflammatory factors such as TNF-α, IL-1β and IL-6 in thetreated groups were similar to those in control groups (FIG. 38 ).

Discussion

Pancreatic beta-cells precisely sense blood glucose fluctuations and inturn dynamically secret insulin to maintain normoglycemia. Generatingartificial cells using synthetic materials to mimic thisglucose-responsive biological process in a robust manner holdstremendous promise for improving outcomes in diabetic patients.Disclosed herein is the construction of an artificial beta-cell (AβC)with a multicompartmental ‘vesicles-in-vesicle’ superstructure that isspatially equipped with a glucose metabolism system and membrane fusionmachinery. Based on the sequential cascade of the glucose uptake,enzymatic oxidation and proton efflux, the AβCs can effectivelydistinguish between high and normal glucose levels via exhibitingdifferent pH values inside the compartment of outer large liposomalvesicles. In hyperglycemic conditions, increased glucose uptake andoxidation generate a low pH (<5.6) which then induces steric deshieldingof peptides tethered to the insulin-loaded inner small liposomalvesicles, such that the peptides on the small vesicles form coiled-coilswith the complementary peptides anchored on the inner surface of largevesicles, subsequently bringing the membranes of the inner and outervesicles together and triggering their fusion to ‘exocytose’ insulin.AβCs transplanted into chemically induced type 1 diabetic mouse modelrestored blood glucose levels to a normal range for at least five days,demonstrating therapeutic potential for treating diabetes.

The presently disclosed AβCs are next generation artificial cells.Different from previously reported single-compartmentalized artificialassemblies, which passively interact with biological systems, ormulticompartmentalized structures, which mimic the hierarchicalarchitecture of cells, the artificial beta-cells disclosed herein areself-regulated, which can effectively sense external changes in glucoselevels, process internal ‘signal transduction’ and ‘exocytose’ insulinas a feedback. Of particular significance, by spatially equipping thevesicles-in-vesicle superstructures with a medically relevant enzymaticcascade system and membrane fusion machinery, the synthetic artificialcells disclosed herein are the first which recreate thestimuli-responsive vesicle fusion-mediated ‘exocytosis’ process, whichfurther guides the evolution of synthetic cells. Such artificial cellswill provide new models to study other biological processes. Moreover,the self-regulated design principle can also be applied for designingother, more complicated artificial cells for replacement of other celldeficiencies such as neurological diseases, and immunological disorders.

The AβCs are novel treatments for diseases such as diabetes. Based onrational design, the AβC precisely controlled blood glucose levels in anormal range for a long term. More importantly, it efficiently avoided apotential risk to hypoglycemia compared to previous pH-sensitivematerials. To date, most reported glucose oxidase-basedglucose-responsive insulin delivery systems mainly utilized matricesconsisting of pH-sensitive materials, which can be eitherswelling/shrinking or degraded under hyperglycemic status in anuncontrollable way once induced. In the present AβCs, protons generatedby glucose oxidation are the signal mediator to induce PEG shielddetachment from insulin-loaded ISVs and made the peptide on ISVssterically unshielded. After that, random collision of ISVs with theouter membrane to induce peptide interactions sustained a low level ofthe fusion process for several hours and avoided hypoglycemia. Byconfining all biochemical processes in well-isolated microenvironmentsdelineated by lipids, fast responsiveness was achieved both in vitro andin vivo. Moreover, based on the reversible nature of pH-tuned PEGattachment and detachment, pulsatile insulin release in response tograded glucose concentrations could run for numerous cycles, therebywithstanding blood glucose fluctuations that are significant features ofeven well-controlled type 1 and type 2 diabetes.

The AβCs are biocompatible. Owning to host rejection of transplantedcells and the extensive immunosuppressive therapy needed to address it,and limited amounts of donor cells, the clinical application ofbeta-cell transplantation has been limited. In contrast, the artificialbeta-cells are easily fabricated in labs and simply ‘transplanted’subcutaneously within e.g., a thermogel, thereby avoiding the use ofimmunosuppressive drug. Due to the biocompatible and biodegradableproperties of the materials used, all the transplanted components werecompletely degraded within 4 weeks with no noticeable inflammatoryregion or fibrotic encapsulation.

Synthetic AβCs recapitulate the key functions of β cells, includingsensing glucose levels, internally transducing signals and dynamicallysecreting insulin via vesicle fusion. Several differences exist betweenAβCs and natural β cells. Natural β cells are electrically excitable andleverage variations in membrane potential to couple fluctuations inblood glucose levels to stimulation or inhibition of insulin secretion.In contrast, the glucose sensing and stimulated insulin exocytosisinside AβCs are simplified into pH-tuned dynamic processes withinconfined microenvironments. Furthermore, natural β cells secrete insulinvia a biphasic process (a rapid first phase and prolonged second phase)to maintain a baseline insulin release at all times. In AβCs, the randomcollision of the insulin-containing ISVs with the outer membrane toinduce peptide interactions made a low level of the fusion processsustainable for several hours. Related to this, the AβCs do not showburst insulin release that has been reported in conventionalpH-responsive materials, conferring protection against potentiallylife-threatening insulin-induced hypoglycemia. Moreover, based on thereversible nature of pH-tuned PEG attachment and detachment, pulsatileinsulin release in response to graded glucose concentrations can run fornumerous cycles, thereby withstanding the blood glucose fluctuationsthat are significant features of even well-controlled type 1 and type 2diabetes. One of the greatest advantages of the AβCs is that they can be‘transplanted’ directly within e.g., an injectable gel or potentialtranscutaneous microneedle patches to restore blood glucose homeostasis,thereby avoiding use of the immunosuppressive drugs required for livecells transplant. Results presented here show that automated dynamiccontrol of blood glucose concentrations to the near-normal range isfeasible with synthetic artificial cells.

From a broad view, by loading different functional proteins or hormonesinsides the ISLs, synthetic cells for treating different cellularfunctional deficiencies can be readily fabricated. Also, by changing theglucose metabolism enzymes inside the OLV with other enzymes orenzymatic systems having different disease-related signal molecules assubstrates, diverse synthetic cells can be obtained for curing differentdiseases. Moreover, besides utilizing pH-responsive DNA bridges tocontrol the anchoring and detachment of the PEG shield, otherstimulus-responsive functional groups that can respond to light,temperature or magnetic field can also be introduced into this system tobuild smart synthetic cells. In addition to utilizing coiled-coilformation peptides to trigger membrane fusion, other mechanisms such ashost-guest supramolecular interaction, zipper DNA duplex formation, andelectrostatic interaction can be employed to pull the membranes of ISVand OLV close together and promote membrane fusion.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations that fall withinthe spirit and scope of the invention.

We claim:
 1. A method of delivering a therapeutic agent to a subjectcomprising: a. providing a particle comprising: an inner liposomalvesicle (ILV) encapsulating insulin or a biologically active compoundderived from insulin; an outer liposomal vesicle (OLV) encapsulating theILV; a membrane fusion-promoting agent, wherein said membranefusion-promoting agent selectively facilitates membrane fusion inresponse to changes in pH within the particle; and a pH-altering agent,wherein said pH-altering agent is a glucose-responsive enzyme inside theOLV but not inside an ILV; b. triggering ILV and OLV fusion; and c.releasing the insulin or biologically active compound derived frominsulin outside of the OLV.
 2. The method of claim 1, wherein themembrane fusion-promoting agent comprises a SNARE polypeptide, aPeptide-K, a Peptide-E, or combinations thereof.
 3. The method of claim1, wherein the membrane fusion-promoting agent comprises a firstmembrane fusion-promoting agent attached to the OLV and a secondmembrane fusion-promoting agent attached to the ILV.
 4. The method ofclaim 1, wherein the glucose-responsive enzyme comprises a glucoseoxidase.
 5. The method of claim 1, wherein the OLV further comprises aglucose membrane transporter.
 6. The method of claim 5, wherein theglucose membrane transporter transports glucose into the OLV.
 7. Themethod of claim 5, wherein the glucose membrane transporter comprises aGlucose Transporter 2 (GLUT2) polypeptide.
 8. The method of claim 1,wherein the OLV further comprises a proton transporter.
 9. The method ofclaim 8, wherein the proton transporter comprises a Gramicidin Apolypeptide.
 10. The method of claim 1, wherein the particle furthercomprises a peroxide-metabolizing enzyme.
 11. The method of claim 10,wherein the peroxide-metabolizing enzyme comprises a catalase (CAT)polypeptide.
 12. The method of claim 1, wherein the ILV furthercomprises a membrane fusion-inhibiting agent which shields access to themembrane fusion-promoting agent.
 13. The method of claim 12, wherein themembrane fusion-inhibiting agent comprises a polyethylene glycol (PEG)molecule.
 14. The method of claim 13, wherein the polyethylene glycol(PEG) molecule comprises an acid-degradable polyethylene glycol (PEG)molecule.
 15. The method of claim 1, further comprising inhibitingILV-OLV fusion by blocking access to the membrane fusion-promotingagent.
 16. The method of claim 1, further comprising oxidizing glucosein the particle.
 17. The method of claim 16, wherein the oxidizing stepreduces the pH of the OLV.
 18. The method of claim 17 wherein thereduced pH of the OLV deshields the membrane fusion-promoting agent. 19.The method of claim 1, wherein the triggering step b) is facilitated bybinding of the membrane fusion-promoting agent.
 20. The method of claim1, wherein the triggering step b) is preceded by binding of a firstmembrane fusion-promoting agent attached to the OLV to a second membranefusion-promoting agent attached to the ILV.